Antisense nucleic acids

ABSTRACT

The present invention provides a pharmaceutical agent which causes skipping of the 55th, 45th, 50th or 44th exon in the human dystrophin gene with a high efficiency. 
     The present invention provides an oligomer which efficiently enables to cause skipping of the 55th, 45th, 50th or 44th exon in the human dystrophin gene.

TECHNICAL FIELD

The present invention relates to an antisense oligomer which causesskipping of exon 55, 45, 50 or 44 in the human dystrophin gene, and apharmaceutical composition comprising the oligomer.

BACKGROUND ART

Duchenne muscular dystrophy (DMD) is the most frequent form ofhereditary progressive muscular dystrophy that affects one in about3,500 newborn boys. Although the motor functions are rarely differentfrom healthy humans in infancy and childhood, muscle weakness isobserved in children from around 4 to 5 years old. Then, muscle weaknessprogresses to the loss of ambulation by about 12 years old and death dueto cardiac or respiratory insufficiency in the twenties. DMD is such asevere disorder. At present, there is no effective therapy for DMDavailable, and it has been strongly desired to develop a noveltherapeutic agent.

DMD is known to be caused by a mutation in the dystrophin gene. Thedystrophin gene is located on X chromosome and is a huge gene consistingof 2.2 million DNA nucleotide pairs. DNA is transcribed into mRNAprecursors, and introns are removed by splicing to synthesize mRNA inwhich 79 exons are joined together. This mRNA is translated into 3,685amino acids to produce the dystrophin protein. The dystrophin protein isassociated with the maintenance of membrane stability in muscle cellsand necessary to make muscle cells less fragile. The dystrophin genefrom patients with DMD contains a mutation and hence, the dystrophinprotein, which is functional in muscle cells, is rarely expressed.Therefore, the structure of muscle cells cannot be maintained in thebody of the patients with DMD, leading to a large influx of calcium ionsinto muscle cells. Consequently, an inflammation-like response occurs topromote fibrosis so that muscle cells can be regenerated only withdifficulty.

Becker muscular dystrophy (BMD) is also caused by a mutation in thedystrophin gene. The symptoms involve muscle weakness accompanied byatrophy of muscle but are typically mild and slow in the progress ofmuscle weakness, when compared to DMD. In many cases, its onset is inadulthood. Differences in clinical symptoms between DMD and BMD areconsidered to reside in whether the reading frame for amino acids on thetranslation of dystrophin mRNA into the dystrophin protein is disruptedby the mutation or not (Non-Patent Document 1). More specifically, inDMD, the presence of mutation shifts the amino acid reading frame sothat the expression of functional dystrophin protein is abolished,whereas in BMD the dystrophin protein that functions, thoughimperfectly, is produced because the amino acid reading frame ispreserved, while a part of the exons are deleted by the mutation.

Exon skipping is expected to serve as a method for treating DMD. Thismethod involves modifying splicing to restore the amino acid readingframe of dystrophin mRNA and induce expression of the dystrophin proteinhaving the function partially restored (Non-Patent Document 2). Theamino acid sequence part, which is a target for exon skipping, will belost. For this reason, the dystrophin protein expressed by thistreatment becomes shorter than normal one but since the amino acidreading frame is maintained, the function to stabilize muscle cells ispartially retained. Consequently, it is expected that exon skipping willlead DMD to the similar symptoms to that of BMD which is milder. Theexon skipping approach has passed the animal tests using mice or dogsand now is currently assessed in clinical trials on human DMD patients.

The skipping of an exon can be induced by binding of antisense nucleicacids targeting either 5′ or 3′ splice site or both sites, orexon-internal sites. An exon will only be included in the mRNA when bothsplice sites thereof are recognized by the spliceosome complex. Thus,exon skipping can be induced by targeting the splice sites withantisense nucleic acids. Furthermore, the binding of an SR protein to anexonic splicing enhancer (ESE) is considered necessary for an exon to berecognized by the splicing mechanism. Accordingly, exon skipping canalso be induced by targeting ESE.

Since a mutation of the dystrophin gene may vary depending on DMDpatients, antisense nucleic acids need to be designed based on the siteor type of respective genetic mutation. In the past, antisense nucleicacids that induce exon skipping for all 79 exons were produced by SteveWilton, et al., University of Western Australia (Non-Patent Document 3),and the antisense nucleic acids which induce exon skipping for 39 exonswere produced by Annemieke Aartsma-Rus, et al., Netherlands (Non-PatentDocument 4).

It is considered that approximately 20% of all DMD patients may betreated by skipping the 55th, the 45th, the 50th and the 44th exons(hereinafter referred to as “exon 55”, “exon 45”, “exon 50” and “exon44”, respectively). In recent years, several research organizationsreported on the studies where exon 55, 45, 50 or 44 in the dystrophingene was targeted for exon skipping (Patent Documents 1 to 8). However,a technique for skipping exon 55, 45, 50 or 44 with a high efficiencyhas not yet been established.

-   Patent Document 1: International Publication WO 2006/000057-   Patent Document 2: International Publication WO 2004/048570-   Patent Document 3: US Unexamined Patent Application Publication US    2010/0168212-   Patent Document 4: International Publication WO2010/048586-   Patent Document 5: International Publication WO 2004/083446-   Patent Document 6: International Publication WO 2010/050801-   Patent Document 7: International Publication WO 2009/139630-   Non-Patent Document 1: Monaco A. P. et al., Genomics 1988; 2: p.    90-95-   Non-Patent Document 2: Matsuo M., Brain Dev 1996; 18: p. 167-172-   Non-Patent Document 3: Wilton S. D., et al., Molecular Therapy 2007:    15: p. 1288-96-   Non-Patent Document 4: Annemieke Aartsma-Rus et al. (2002)    Neuromuscular Disorders 12: S71-S77-   Non-Patent Document 5: Linda J. Popplewell et al., (2010)    Neuromuscular Disorders, vol. 20, no. 2, p. 102-10

DISCLOSURE OF THE INVENTION

Under the foregoing circumstances, antisense oligomers that stronglyinduce skipping of exon 55, exon 45, exon 50 or exon 44 in thedystrophin gene and muscular dystrophy therapeutics comprising oligomersthereof have been desired.

As a result of detailed studies of the structure of the dystrophin gene,the present inventors have found that exon 55 skipping can be inducedwith a high efficiency by antisense oligomers which target the sequenceconsisting of around the 1st to the 21st, the 11th to the 31st, and the14th to the 34th nucleotides from the 5′ end of exon 55 in the mRNAprecursor (hereinafter referred to as “pre-mRNA”) in the dystrophin genewith antisense oligomers.

The present inventors have also found that exon 45 skipping can beinduced with a high efficiency by antisense oligomers which target thesequence consisting of around the 1st to the 25th and the 6th to the30th nucleotides from the 5′ end of exon 45 in the pre-mRNA in thedystrophin gene with antisense oligomers.

Furthermore, the present inventors have found that exon 50 skipping canbe induced with a high efficiency by antisense oligomers which targetthe sequence consisting of around the 107th to the 127th nucleotidesfrom the 5′ end of exon 50 in the pre-mRNA in the dystrophin gene withantisense oligomers.

Additionally, the present inventors have also found that exon 44skipping can be induced with a high efficiency by antisense oligomerswhich target the sequence consisting of around the 11th to the 32nd andthe 26th to the 47th nucleotides from the 5′ end of exon 44 in thepre-mRNA in the dystrophin gene with antisense oligomers.

Based on this finding, the present inventors have accomplished thepresent invention.

That is, the present invention is as follows.

[1] An antisense oligomer which causes skipping of the 55th exon in thehuman dystrophin gene, consisting of a nucleotide sequence complementaryto any one of the nucleotide sequences consisting of the −2nd to the19th, the −2nd to the 20th, the −2nd to the 21st, the −2nd to the 22nd,the −2nd to the 23rd, the −1st to the 19th, the −1st to the 20th, the−1st to the 21st, the −1st to the 22nd, the −1st to the 23rd, the 1st tothe 19th, the 1st to the 20th, the 1st to the 21st, the 1st to the 22nd,the 1st to the 23rd, the 2nd to the 19th, the 2nd to the 20th, the 2ndto the 21st, the 2nd to the 22nd, the 2nd to the 23rd, the 3rd to the19th, the 3rd to the 20th, the 3rd to the 21st, the 3rd to the 22nd, the3rd to the 23rd, the 9th to the 29th, the 9th to the 30th, the 9th tothe 31st, the 9th to the 32nd, the 9th to the 33rd, the 10th to the29th, the 10th to the 30th, the 10th to the 31st, the 10th to the 32nd,the 10th to the 33rd, the 11th to the 29th, the 11th to the 30th, the11th to the 31st, the 11th to the 32nd, the 11th to the 33rd, the 12thto the 29th, the 12th to the 30th, the 12th to the 31st, the 12th to the32nd, the 12th to the 33rd, the 13th to the 29th, the 13th to the 30th,the 13th to the 31st, the 13th to the 32nd, the 13th to the 33rd, the12th to the 34th, the 12th to the 35th, the 12th to the 36th, the 13thto the 34th, the 13th to the 35th, the 13th to the 36th, the 14th to the32nd, the 14th to the 33rd, the 14th to the 34th, 14th to the 35th, the14th to the 36th, the 15th to the 32nd, the 15th to the 33rd, the 15thto the 34th, the 15th to the 35th, the 15th to the 36th, the 16th to the32nd, the 16th to the 33rd, the 16th to the 34th, the 16th to the 35th,or the 16th to the 36th nucleotides, from the 5′ end of the 55th exon inthe human dystrophin gene.

[2] An antisense oligomer which causes skipping of the 45th exon in thehuman dystrophin gene, consisting of a nucleotide sequence complementaryto any one of the nucleotide sequences consisting of the −3rd to the19th, the −3rd to the 20th, the −3rd to the 21st, the −3 rd to the 22nd,the −3rd to the 23rd, the −2nd to the 19th, the −2nd to the 20th, the−2nd to the 21st, the −2nd to the 22nd, the −2nd to the 23rd, the −1stto the 19th, the −1st to the 20th, the −1 st to the 21st, the −1st tothe 22nd, the −1st to the 23rd, the 1st to the 19th, the 1st to the20th, the 1st to the 21st, the 1st to the 22nd, the 1st to the 23rd, the2nd to the 19th, the 2nd to the 20th, the 2nd to the 21st, the 2nd tothe 22nd, the 2nd to the 23rd, the −2nd to the 24th, the −2nd to the25th, the −2nd to the 26th, the −2 nd to the 27th, the −1st to the 24th,the −1st to the 25th, the −1st to the 26th, the −1st to the 27th, the1st to the 24th, the 1st to the 25th, the 1st to the 26th, the 1st tothe 27th, the 2nd to the 24th, the 2nd to the 25th, the 2nd to the 26th,the 2nd to the 27th, the 3rd to the 23rd, the 3rd to the 24th, the 3rdto the 25th, the 3rd to the 26th, the 3rd to the 27th, the 4th to the28th, the 4th to the 29th, the 4th to the 30th, the 4th to the 31st, the4th to the 32nd, the 5th to the 28th, the 5th to the 29th, the 5th tothe 30th, the 5th to the 31st, the 5th to the 32nd, the 6th to the 28th,the 6th to the 29th, the 6th to the 30th, the 6th to the 31st, the 6thto the 32nd, the 7th to the 28th, the 7th to the 29th, the 7th to the30th, the 7th to the 31st, the 7th to the 32nd, the 8th to the 28th, the8th to the 29th, the 8th to the 30th, the 8th to the 31st, or the 8th tothe 32nd nucleotides, from the 5′ end of the 45th exon in the humandystrophin gene.

[3] An antisense oligomer which causes skipping of the 50th exon in thehuman dystrophin gene, consisting of a nucleotide sequence complementaryto any one of the nucleotide sequences consisting of the 105th to the125th, the 105th to the 126th, the 105th to the 127th, the 105th to the128th, the 105th to the 129th, the 106th to the 125th, the 106th to the126th, the 106th to the 127th, the 106th to the 128th, the 106th to the129th, the 107th to the 125th, the 107th to the 126th, the 107th to the127th, the 107th to the 128th, the 107th to the 129th, the 108th to the125th, the 108th to the 126th, the 108th to the 127th, the 108th to the128th, the 108th to the 129th, the 109th to the 125th, the 109th to the126th, the 109th to the 127th, the 109th to the 128th, or the 109th tothe 129th nucleotides, from the 5′ end of the 50th exon in the humandystrophin gene.

[4] An antisense oligomer which causes skipping of the 44th exon in thehuman dystrophin gene, consisting of a nucleotide sequence complementaryto any one of the nucleotide sequences consisting of the 9th to the30th, 9th to the 31st, the 9th to the 32nd, the 9th to the 33rd, the 9thto the 34th, the 10th to the 30th, the 10th to the 31st, the 10th to the32nd, the 10th to the 33rd, the 10th to the 34th, the 11th to the 30th,the 11th to the 31st, the 11th to the 32nd, the 11th to the 33rd, the11th to the 34th, the 12th to the 30th, the 12th to the 31st, the 12thto the 32nd, the 12th to the 33rd, the 12th to the 34th, the 13th to the30th, the 13th to the 31st, the 13th to the 32nd, the 13th to the 33rd,the 13th to the 34th, the 24th to the 45th, the 24th to the 46th, the24th to the 47th, the 24th to the 48th, the 24th to the 49th, the 25thto the 45th, the 25th to the 46th, the 25th to the 47th, the 25th to the48th, the 25th to the 49th, the 26th to the 45th, the 26th to the 46th,the 26th to the 47th, the 26th to the 48th, the 26th to the 49th, the27th to the 45th, the 27th to the 46th, the 27th to the 47th, the 27thto the 48th, the 27th to the 49th, the 28th to the 45th, the 28th to the46th, the 28th to the 47th, the 28th to the 48th, the 28th to the 49th,the 29th to the 45th, the 29th to the 46th, the 29th to the 47th, the29th to the 48th, or the 29th to the 49th nucleotides, from the 5′ endof the 44th exon in the human dystrophin gene.

[5] The antisense oligomer according to [1], which consists of acomplementary sequence to the nucleotide sequences consisting of the 1stto the 21st, the 11th to the 31st, or the 14th to the 34th nucleotides,from the 5′ end of the 55th exon in the human dystrophin gene.

[6] The antisense oligomer according to [1], consisting of thenucleotide sequence shown by any one selected from the group consistingof the 170th to the 190th, the 160th to the 180th, and the 157th to the177th nucleotides of SEQ ID NO: 5.

[7] The antisense oligomer according to [2], which consists of acomplementary sequence to the nucleotide sequences consisting of the−2nd to the 19th, the 1st to the 21st, the 1st to the 25th, or the 6thto the 30th nucleotides, from the 5′ end of the 45th exon in the humandystrophin gene.

[8] The antisense oligomer according to [2], consisting of thenucleotide sequence shown by any one selected from the group consistingof the 158th to the 178th, the 156th to the 176th, the 152nd to the176th, and the 147th to the 171st nucleotides of SEQ ID NO: 6.

[9] The antisense oligomer according to [3], which consists of acomplementary sequence to the nucleotide sequences consisting of the106th to the 126th or the 107th to the 127th nucleotides, from the 5′end of the 50th exon in the human dystrophin gene.

[10] The antisense oligomer according to [3], consisting of thenucleotide sequence shown by any one selected from the group consistingof the 4th to the 24th and the 3rd to the 23rd nucleotides of SEQ ID NO:7.

[11] The antisense oligomer according to [4], which consists of acomplementary sequence to the nucleotide sequences consisting of the11th to the 32nd, the 25th to the 45th, the 26th to the 46th, the 26thto the 47th or the 27th to the 47th nucleotides, from the 5′ end of the44th exon in the human dystrophin gene.

[12] The antisense oligomer according to [4], consisting of thenucleotide sequence shown by any one selected from the group consistingof the 117th to the 138th, the 104th to the 124th, the 103rd to the123rd, the 102nd to the 123rd and the 102nd to the 122nd nucleotides ofSEQ ID NO: 8.

[13] The antisense oligomer according to any one of [1] to [12], whichis an oligonucleotide.

[14] The antisense oligomer according to [13], wherein the sugar moietyand/or the phosphate-binding region of at least one nucleotideconstituting the oligonucleotide is modified.

[15] The antisense oligomer according to [14], wherein the sugar moietyof at least one nucleotide constituting the oligonucleotide is a ribosein which the 2′-OH group is replaced by any one selected from the groupconsisting of OR, R, R′OR, SH, SR, NH₂, NHR, NR₂, N₃, CN, F, Cl, Br andI (wherein R is an alkyl or an aryl and R′ is an alkylene).

[16] The antisense oligomer according to [14] or [15], wherein thephosphate-binding region of at least one nucleotide constituting theoligonucleotide is any one selected from the group consisting of aphosphorothioate bond, a phosphorodithioate bond, an alkylphosphonatebond, a phosphoramidate bond and a boranophosphate bond.

[17] The antisense oligomer according to any one of [1] to [12], whichis a morpholino oligomer.

[18] The antisense oligomer according to [17], which is aphosphorodiamidate morpholino oligomer.

[19] The antisense oligomer according to [17] or [18], wherein the 5′end is any one of the groups of chemical formulae (1) to (3) below:

[20] A pharmaceutical composition for the treatment of musculardystrophy, comprising as an active ingredient the antisense oligomeraccording to any one of [1] to [19], or a pharmaceutically acceptablesalt or hydrate thereof.

The antisense oligomer of the present invention can induce skipping ofexon 55, exon 45, exon 50 or exon 44 in the human dystrophin gene withhigh efficiencies. Also, the symptoms of Duchenne muscular dystrophy canbe effectively alleviated by administering the pharmaceuticalcomposition of the present invention. In addition, since the antisenseoligomer of the present invention targets only exon sequences inpatients, the target sequences are conserved among individuals comparedto the cases with those targeting sequences in introns. Therefore, theantisense oligomer of the present invention is capable of achievingexcellent skipping efficiencies regardless of individual varieties(personal differences). Furthermore, the antisense oligomer of thepresent invention has short length of 20 bp or around and has lessprobability of containing mutations raised from individual varieties(interpersonality) e.g. SNP (Single Nucleotide Polymorphism) in thetarget sequences compared to conventional antisense oligomers for DMDtreatment having lengths of 25 bp or so. This feature also helps theantisense oligomer of the present invention in achieving excellentskipping efficiencies regardless of individual variety (personaldifferences). Moreover, the antisense oligomer of the present inventionhave less side effects raised by the induction of cytokines and so on,since antisense oligomers having shorter chains have less tendency toinduce immunity in general.

Also, since the antisense oligomer of the present invention is rathershort, the cost of manufacturing is relatively small.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the efficiency of exon 45 skipping by 2′-OMe-S-RNA oligomerin the human dystrophin gene in human rhabdomyosarcoma cell line (RDcells).

FIG. 2 shows the efficiency of exon 45 skipping by 2′-OMe-S-RNA oligomerin the human dystrophin gene in human rhabdomyosarcoma cell line (RDcells).

FIG. 3 shows the efficiency of exon 45 skipping by PMO in the humandystrophin gene in human rhabdomyosarcoma cell line (RD cells).

FIG. 4 shows the efficiency of exon 45 skipping by PMO in the humandystrophin gene in human rhabdomyosarcoma cell line (RD cells).

FIG. 5 shows the efficiency of exon 45 skipping by PMO in the humandystrophin gene in the cells where human MyoD gene is induced intofibroblasts from human DMD patient (GM05017 cells) to inducedifferentiation into muscle cells.

FIG. 6 shows the efficiency of exon 55 skipping by 2′-OMe-S-RNA oligomerin the human dystrophin gene in human rhabdomyosarcoma cell line (RDcells).

FIG. 7 shows the efficiency of exon 55 skipping by 2′-OMe-S-RNA oligomerin the human dystrophin gene in human rhabdomyosarcoma cell line (RDcells).

FIG. 8 shows the efficiency of exon 55 skipping by PMO in the humandystrophin gene in human rhabdomyosarcoma cell line (RD cells).

FIG. 9 shows the efficiency of exon 44 skipping by 2′-OMe-S-RNA oligomerin the human dystrophin gene in human rhabdomyosarcoma cell line (RDcells).

FIG. 10 shows the efficiency of exon 44 skipping by 2′-OMe-S-RNAoligomer in the human dystrophin gene in human rhabdomyosarcoma cellline (RD cells).

FIG. 11 shows the efficiency of exon 44 skipping by PMO in the humandystrophin gene in human rhabdomyosarcoma cell line (RD cells).

FIG. 12 shows the efficiency of exon 44 skipping by PMO in the humandystrophin gene in human rhabdomyosarcoma cell line (RD cells).

FIG. 13 shows the efficiency of exon 50 skipping by PMO in the humandystrophin gene in human rhabdomyosarcoma cell line (RD cells).

FIG. 14 shows the efficiency of exon 45 skipping by PMO in the humandystrophin gene in human rhabdomyosarcoma cell line (RD cells).

FIG. 15 shows the efficiency of exon 45 skipping by PMO in the humandystrophin gene in human rhabdomyosarcoma cell line (RD cells).

FIG. 16 shows the efficiency of exon 55 skipping by PMO in the humandystrophin gene in human rhabdomyosarcoma cell line (RD cells).

FIG. 17 shows the efficiency of exon 55 skipping by PMO in the humandystrophin gene in human rhabdomyosarcoma cell line (RD cells).

FIG. 18 shows the efficiency of exon 44 skipping by PMO in the humandystrophin gene in human rhabdomyosarcoma cell line (RD cells).

FIG. 19 shows the efficiency of exon 50 skipping by PMO in the humandystrophin gene in human rhabdomyosarcoma cell line (RD cells).

FIG. 20 shows the efficiency of exon 44 skipping by PMO in the humandystrophin gene in the fibroblasts from human DMD patient with deletionof exon 45 (GM05112 cells).

FIG. 21 shows the effect (Western Blotting) of exon 44 skipping by PMOin the human dystrophin gene in the fibroblasts from human DMD patientwith deletion of exon 45 (GM05112 cells).

FIG. 22 shows the effect (RT-PCR) of exon 50 skipping by PMO in thehuman dystrophin gene in the fibroblasts from human DMD patient withdeletion of exon 45 (GM05112 cells).

FIG. 23 shows the efficiency of exon 50 skipping by PMO in the humandystrophin gene in the fibroblasts from human DMD patient with deletionof exon 45 (GM05112 cells).

FIG. 24 shows the effect (RT-PCR) of exon 55 skipping by PMO in thehuman dystrophin gene in the fibroblasts from human DMD patient withdeletion of exon 45 (GM05112 cells).

FIG. 25 shows the efficiency of exon 55 skipping by PMO in the humandystrophin gene in the fibroblasts from human DMD patient with deletionof exon 45 (GM05112 cells).

FIG. 26 shows the effect (RT-PCR) of exon 50 skipping by PMO in thehuman dystrophin gene in the fibroblasts from human DMD patient withduplication of exons 8-9 (11-0627 cells).

FIG. 27 shows the efficiency of exon 50 skipping by PMO in the humandystrophin gene in the fibroblasts from human DMD patient withduplication of exons 8-9 (11-0627 cells).

FIG. 28 shows the effect (RT-PCR) of exon 50 skipping by PMO in thehuman dystrophin gene in the fibroblasts from human DMD patient withdeletion of exons 51-55 (GM04364 cells).

FIG. 29 shows the efficiency of exon 50 skipping by PMO in the humandystrophin gene in the fibroblasts from human DMD patient with deletionof exons 51-55 (GM04364 cells).

FIG. 30 shows the effect (RT-PCR) of exon 55 skipping by PMO in thehuman dystrophin gene in the fibroblasts from human DMD patient withdeletion of exon 54 (04-035 cells).

FIG. 31 shows the efficiency of exon 55 skipping by PMO in the humandystrophin gene in the fibroblasts from human DMD patient with deletionof exon 54 (04-035 cells).

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in detail. Theembodiments described below are intended to be presented by way ofexample merely to describe the invention but not limited only to thefollowing embodiments. The present invention may be implemented invarious ways without departing from the gist of the invention.

All of the publications, published patent applications, patents andother patent documents cited in the specification are hereinincorporated by reference in their entirety. The specification herebyincorporates by reference the contents of the specification and drawingsin the Japanese Patent Application (No. 2011-288040) filed Dec. 28, 2011and the Japanese Patent Application (No. 2012-043092) filed Feb. 29,2012, from which the priority was claimed.

Hereinafter, the present invention is described in detail. Theembodiments described below are intended to be presented by way ofexample merely to describe the invention but not limited only to thefollowing embodiments. The present invention may be implemented invarious ways without departing from the gist of the invention.

Without description in particular, the amino acid sequence representsthe amino terminus as left and carboxyl terminus as right, and the basesequence represents the 5′ end as left and the 3′ end as right.

1. Antisense Oligomer

The present invention provides the antisense oligomer (hereinafterreferred to as the “exon 55 skipping oligomer of the present invention”)which causes skipping of exon 55 in the human dystrophin gene,consisting of a nucleotide sequence complementary to any one of thenucleotide sequences (hereinafter also referred to as the “exon 55target sequence”) consisting of the −2nd to the 19th, the −2nd to the20th, the −2nd to the 21st, the −2nd to the 22nd, the −2nd to the 23rd,the −1st to the 19th, the −1st to the 20th, the −1st to the 21st, the−1st to the 22nd, the −1st to the 23rd, the 1st to the 19th, the 1st tothe 20th, the 1st to the 21st, the 1st to the 22nd, the 1st to the 23rd,the 2nd to the 19th, the 2nd to the 20th, the 2nd to the 21st, the 2ndto the 22nd, the 2nd to the 23rd, the 3rd to the 19th, the 3rd to the20th, the 3rd to the 21st, the 3rd to the 22nd, the 3rd to the 23rd, the9th to the 29th, the 9th to the 30th, the 9th to the 31st, the 9th tothe 32nd, the 9th to the 33rd, the 10th to the 29th, the 10th to the30th, the 10th to the 31st, the 10th to the 32nd, the 10th to the 33rd,the 11th to the 29th, the 11th to the 30th, the 11th to the 31st, the11th to the 32nd, the 11th to the 33rd, the 12th to the 29th, the 12thto the 30th, the 12th to the 31st, the 12th to the 32nd, the 12th to the33rd, the 13th to the 29th, the 13th to the 30th, the 13th to the 31st,the 13th to the 32nd, the 13th to the 33rd, the 12th to the 34th, the12th to the 35th, the 12th to the 36th, the 13th to the 34th, the 13thto the 35th, the 13th to the 36th, the 14th to the 32nd, the 14th to the33rd, the 14th to the 34th, the 14th to the 35th, the 14th to the 36th,the 15th to the 32nd, the 15th to the 33rd, the 15th to the 34th, the15th to the 35th, the 15th to the 36th, the 16th to the 32nd, the 16thto the 33rd, the 16th to the 34th, the 16th to the 35th, or the 16th tothe 36th nucleotides, from the 5′ end of exon 55 in the human dystrophingene.

The present invention also provides the antisense oligomer (hereinafterreferred to as the “exon 45 skipping oligomer of the present invention”)which causes skipping of exon 45 in the human dystrophin gene,consisting of a nucleotide sequence complementary to any one of thenucleotide sequences (hereinafter also referred to as the “exon 45target sequence”) consisting of the −3rd to the 19th, the −3rd to the20th, the −3rd to the 21st, the −3rd to the 22nd, the −3rd to the 23rd,the −2nd to the 19th, the −2nd to the 20th, the −2nd to the 21st, the−2nd to the 22nd, the −2nd to the 23rd, the −1st to the 19th, the −1stto the 20th, the −1st to the 21st, the −1st to the 22nd, the −1st to the23rd, the 1st to the 19th, the 1st to the 20th, the 1st to the 21st, the1st to the 22th, the 1st to the 23rd, the 2nd to the 19th, the 2nd tothe 20th, the 2nd to the 21st, the 2nd to the 22nd, the 2nd to the 23rd,the −2nd to the 24th, the −2nd to the 25th, the −2nd to the 26th, the−2nd to the 27th, the −1st to the 24th, the −1st to the 25th, the −1stto the 26th, the −1st to the 27th, the 1st to the 24th, the 1st to the25th, the 1st to the 26th, the 1st to the 27th, the 2nd to the 24th, the2nd to the 25th, the 2nd to the 26th, the 2nd to the 27th, the 3rd tothe 23rd, the 3rd to the 24th, the 3rd to the 25th, the 3rd to the 26th,the 3rd to the 27th, the 4th to the 28th, the 4th to the 29th, the 4thto the 30th, the 4th to the 31th, the 4th to the 32nd, the 5th to the28th, the 5th to the 29th, the 5th to the 30th, the 5th to the 31st, the5th to the 32nd, the 6th to the 28th, the 6th to the 29th, the 6th tothe 30th, the 6th to the 31st, the 6th to the 32nd, the 7th to the 28th,the 7th to the 29th, the 7th to the 30th, the 7th to the 31st, the 7thto the 32nd, the 8th to the 28th, the 8th to the 29th, the 8th to the30th, the 8th to the 31st, or the 8th to the 32nd nucleotides, from the5′ end of exon 45 in the human dystrophin gene.

Additionally, the present invention provides the antisense oligomer(hereinafter referred to as the “exon 50 skipping oligomer of thepresent invention”) which causes skipping of exon 50 in the humandystrophin gene, consisting of a nucleotide sequence complementary toany one of the nucleotide sequences (hereinafter also referred to as the“exon 50 target sequence”) consisting of the 105th to the 125th, the105th to the 126th, the 105th to the 127th, the 105th to the 128th, the105th to the 129th, the 106th to the 125th, the 106th to the 126th, the106th to the 127th, the 106th to the 128th, the 106th to the 129th, the107th to the 125th, the 107th to the 126th, the 107th to the 127th, the107th to the 128th, the 107th to the 129th, the 108th to the 125th, the108th to the 126th, the 108th to the 127th, the 108th to the 128th, the108th to the 129th, the 109th to the 125th, the 109th to the 126th, the109th to the 127th, the 109th to the 128th, or the 109th to the 129thnucleotides, from the 5′ end of exon 50 in the human dystrophin gene.

Furthermore, the present invention provides the antisense oligomer(hereinafter referred to as the “exon 44 skipping oligomer of thepresent invention”) which causes skipping of exon 44 in the humandystrophin gene, consisting of a nucleotide sequence complementary toany one of the nucleotide sequences (hereinafter also referred to as the“exon 44 target sequence”) consisting of the 9th to the 30th, the 9th tothe 31st, the 9th to the 32nd, the 9th to the 33rd, the 9th to the 34th,the 10th to the 30th, the 10th to the 31st, the 10th to the 32nd, the10th to the 33rd, the 10th to the 34th, the 11th to the 30th, the 11thto the 31st, the 11th to the 32nd, the 11th to the 33rd, the 11th to the34th, the 12th to the 30th, the 12th to the 31st, the 12th to the 32nd,the 12th to the 33rd, the 12th to the 34th, the 13th to the 30th, the13th to the 31st, the 13th to the 32nd, the 13th to the 33rd, the 13thto the 34th, the 24th to the 45th, the 24th to the 46th, the 24th to the47th, the 24th to the 48th, the 24th to the 49th, the 25th to the 45th,the 25th to the 46th, the 25th to the 47th, the 25th to the 48th, the25th to the 49th, the 26th to the 45th, the 26th to the 46th, the 26thto the 47th, the 26th to the 48th, the 26th to the 49th, the 27th to the45th, the 27th to the 46th, the 27th to the 47th, the 27th to the 48th,the 27th to the 49th, the 28th to the 45th, the 28th to the 46th, the28th to the 47th, the 27th to the 48th, the 27th to the 49th, the 28thto the 45th, the 28th to the 46th, the 28th to the 47th, the 28th to the48th, the 28th to the 49th, the 29th to the 45th, the 29th to the 46th,the 29th to the 47th, the 29th to the 48th, or the 29th to the 49thnucleotides, from the 5′ end of exon 44 in the human dystrophin gene.

Hereinafter, the skipping oligomers of exon 55, 45, 50 and 44 may becollectively referred to as the “oligomers of the present invention”.

[Exon 55, 45, 50 and 44 in Human Dystrophin Gene]

In the present invention, the term “gene” is intended to mean a genomicgene and also include cDNA, mRNA precursor and mRNA. Preferably, thegene is mRNA precursor, i.e. pre-mRNA.

In the human genome, the human dystrophin gene locates at locus Xp21.2.The human dystrophin gene has a size of 3.0 Mbp and is the largest geneamong known human genes. However, the coding regions of the humandystrophin gene are only 14 kb, distributed as 79 exons throughout thehuman dystrophin gene (Roberts, R G, et al., Genomics, 16: 536-538(1993)). The pre-mRNA, which is the transcript of the human dystrophingene, undergoes splicing to generate mature mRNA of 14 kb. Thenucleotide sequence of human wild-type dystrophin gene is known(GeneBank Accession No. NM_004006).

The nucleotide sequence consisting of the −2nd to the 190th nucleotides,from the 5′ end of exon 55 in the human wild-type dystrophin gene isrepresented by SEQ ID NO: 1. The nucleotide sequence consisting of the−3rd to the 176th nucleotides, from the 5′ end of exon 45 in the humanwild-type dystrophin gene is represented by SEQ ID NO: 2. The nucleotidesequence consisting of the 1st to the 109th nucleotides, from the 5′ endof exon 50 and the 1st to the 20th nucleotides, from the 5′ end ofintron 50 in the human wild-type dystrophin gene is represented by SEQID NO: 3.

The nucleotide sequence consisting of the 1st to the 148th nucleotides,from the 5′ end of exon 44 in the human wild-type dystrophin gene isrepresented by SEQ ID NO: 4.

The oligomer of the present invention is designed to cause skipping ofexon 55, 45, 50 or 44 in the human dystrophin gene, thereby modifyingthe protein encoded by DMD type of dystrophin gene into the BMD type ofdystrophin protein. Accordingly, exon 55, 45, 50 and 44 in thedystrophin gene that are the targets of exon skipping by the oligomer ofthe present invention include both wild and mutant types.

Specifically, exon 55, 45, 50 and 44 mutants of the human dystrophingene are the polynucleotides defined in (a) or (b) below.

(a) A polynucleotide that hybridizes under stringent conditions to apolynucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 1 (or a nucleotide sequence consistingof the 3rd to the 192nd nucleotides of SEQ ID NO:1), SEQ ID NO: 2 (or anucleotide sequence consisting of the 4th to the 179th nucleotides ofSEQ ID NO:2), SEQ ID NO: 3 (or a nucleotide sequence consisting of the1st to the 109th nucleotides of SEQ ID NO:3), or SEQ ID NO: 4;

(b) A polynucleotide consisting of a nucleotide sequence having at least90% homology with the nucleotide sequence of SEQ ID NO:1 (or anucleotide sequence consisting of the 3rd to the 192nd nucleotides ofSEQ ID NO:1), SEQ ID NO: 2 (or a nucleotide sequence consisting of the4th to the 179th nucleotides of SEQ ID NO:2), SEQ ID NO: 3 (or anucleotide sequence consisting of the 1st to the 109th nucleotides ofSEQ ID NO:3), or SEQ ID NO: 4.

As used herein, the term “polynucleotide” is intended to mean DNA orRNA.

As used herein, the term “polynucleotide that hybridizes under stringentconditions” refers to, for example, a polynucleotide obtained by colonyhybridization, plaque hybridization, Southern hybridization or the like,using as a probe all or part of a polynucleotide consisting of anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO: 1 (or a nucleotide sequence consisting of the 3rd to the 192ndnucleotides of SEQ ID NO:1), SEQ ID NO: 2 (or a nucleotide sequenceconsisting of the 4th to the 179th nucleotides of SEQ ID NO:2), SEQ IDNO: 3 (or a nucleotide sequence consisting of the 1st to the 109thnucleotides of SEQ ID NO:3), or SEQ ID NO: 4. The hybridization methodwhich may be used includes methods described in, for example, “Sambrook& Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold SpringHarbor, Laboratory Press 2001,” “Ausubel, Current Protocols in MolecularBiology, John Wiley & Sons 1987-1997,” etc.

As used herein, the term “complementary nucleotide sequence” is notlimited only to nucleotide sequences that form Watson-Crick pairs withtarget nucleotide sequences, but is intended to also include nucleotidesequences which form Wobble base pairs. As used herein, the termWatson-Crick pair refers to a pair of nucleobases in which hydrogenbonds are formed between adenine-thymine, adenine-uracil orguanine-cytosine, and the term Wobble base pair refers to a pair ofnucleobases in which hydrogen bonds are formed between guanine-uracil,inosine-uracil, inosine-adenine or inosine-cytosine. As used herein, theterm “complementary nucleotide sequence” does not only refers to anucleotide sequence 100% complementary to the target nucleotide sequencebut also refers to a complementary nucleotide sequence that may contain,for example, 1 to 3, 1 to 2, or one nucleotide non-complementary to thetarget nucleotide sequence.

As used herein, the term “stringent conditions” may be any of lowstringent conditions, moderate stringent conditions or high stringentconditions. The term “low stringent condition” is, for example, 5×SSC,5×Denhardt's solution, 0.5% SDS, 50% formamide at 32° C. The term“moderate stringent condition” is, for example, 5×SSC, 5×Denhardt'ssolution, 0.5% SDS, 50% formamide at 42° C., or 5×SSC, 1% SDS, 50 mMTris-HCl (pH 7.5), 50% formamide at 42° C. The term “high stringentcondition” is, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50%formamide at 50° C. or 0.2×SSC, 0.1% SDS at 65° C. Under theseconditions, polynucleotides with higher homology are expected to beobtained efficiently at higher temperatures, although multiple factorsare involved in hybridization stringency including temperature, probeconcentration, probe length, ionic strength, time, salt concentrationand others, and those skilled in the art may approximately select thesefactors to achieve similar stringency.

When commercially available kits are used for hybridization, forexample, an Alkphos Direct Labelling and Detection System (GEHealthcare) may be used. In this case, according to the attachedprotocol, after cultivation with a labeled probe overnight, the membraneis washed with a primary wash buffer containing 0.1% (w/v) SDS at 55°C., thereby detecting hybridized polynucleotides. Alternatively, whenthe probe is labeled with digoxigenin (DIG) using a commerciallyavailable reagent (e.g., a PCR Labelling Mix (Roche Diagnostics), etc.)in producing a probe based on all or part of the complementary sequenceto the nucleotide sequence of SEQ ID NO: 1 (or a nucleotide sequenceconsisting of the 3rd to the 192nd nucleotides of SEQ ID NO:1), SEQ IDNO: 2 (or a nucleotide sequence consisting of the 4th to the 179thnucleotides of SEQ ID NO:2), SEQ ID NO: 3 (or a nucleotide sequenceconsisting of the 1st to the 109th nucleotides of SEQ ID NO:3), or SEQID NO: 4, hybridization can be detected with a DIG Nucleic AcidDetection Kit (Roche Diagnostics).

In addition to the polynucleotides described above, otherpolynucleotides that can be hybridized include polynucleotides having90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% orhigher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99%or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% orhigher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% orhigher, 99.9% or higher identity with the polynucleotide of SEQ ID NO: 1(or a nucleotide sequence consisting of the 3rd to the 192nd nucleotidesof SEQ ID NO:1), SEQ ID NO: 2 (or a nucleotide sequence consisting ofthe 4th to the 179th nucleotides of SEQ ID NO:2), SEQ ID NO: 3 (or anucleotide sequence consisting of the 1st to the 109th nucleotides ofSEQ ID NO:3), or SEQ ID NO: 4, as calculated by homology search softwareBLAST using the default parameters.

The identity between nucleotide sequences may be determined usingalgorithm BLAST (Basic Local Alignment Search Tool) by Karlin andAltschul (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc. Natl.Acad. Sci. USA 90: 5873, 1993). Programs called BLASTN and BLASTX basedon the BLAST algorithm have been developed (Altschul S F, et al: J. Mol.Biol. 215: 403, 1990). When a nucleotide sequence is sequenced usingBLASTN, the parameters are, for example, score=100 and word-length=12.When BLAST and Gapped BLAST programs are used, the default parametersfor each program are employed.

The sequence complementary to the nucleotide sequence consisting of the−2nd to the 190th nucleotides from the 5′ end of exon 55 is representedby SEQ ID NO: 5. Herein, the nucleotide sequence consisting of the −2ndto the −1st nucleotides, from the 5′ end of exon 55 (the nucleotidesequence consisting of the 1st to the 2nd nucleotides of SEQ ID NO: 1)represents nucleotide sequence consisting of 2 nucleotides at the most3′ end downstream of intron 54 which is located between exon 54 and exon55. More specifically, the nucleotide sequence of exon 55 is oneconsisting of the 3rd to the 192nd nucleotides of SEQ ID NO: 1 and thesequence complementary to exon 55 is one consisting of the 1st to the190th nucleotides of SEQ ID NO: 5.

Herein, the complementary sequence to the nucleotide sequencesconsisting of the −2nd to the 19th, the −2nd to the 20th, the −2nd tothe 21st, the −2nd to the 22nd, the −2nd to the 23rd, the −1st to the19th, the −1st to the 20th, the −1 st to the 21st, the −1st to the 22nd,the −1st to the 23rd, the 1st to the 19th, the 1st to the 20th, the 1stto the 21st, the 1st to the 22nd, the 1st to the 23rd, the 2nd to the19th, the 2nd to the 20th, the 2nd to the 21st, the 2nd to the 22nd, the2nd to the 23rd, the 3rd to the 19th, the 3rd to the 20th, the 3rd tothe 21st, the 3rd to the 22nd, the 3rd to the 23rd, the 9th to the 29th,the 9th to the 30th, the 9th to the 31st, the 9th to the 32nd, the 9thto the 33rd, the 10th to the 29th, the 10th to the 30th, the 10th to the31st, the 10th to the 32nd, the 10th to the 33rd, the 11th to the 29th,the 11th to the 30th, the 11th to the 31st, the 11th to the 32nd, the11th to the 33rd, the 12th to the 29th, the 12th to the 30th, the 12thto the 31st, the 12th to the 32nd, the 12th to the 33rd, the 13th to the29th, the 13th to the 30th, the 13th to the 31st, the 13th to the 32nd,the 13th to the 33rd, the 12th to the 34th, the 12th to the 35th, the12th to the 36th, the 13th to the 34th, the 13th to the 35th, the 13thto the 36th, the 14th to the 32nd, the 14th to the 33rd, the 14th to the34th, the 14th to the 35th, the 14th to the 36th, the 15th to the 32nd,the 15th to the 33rd, the 15th to the 34th, the 15th to the 35th, the15th to the 36th, the 16th to the 32nd, the 16th to the 33rd, the 16thto the 34th, the 16th to the 35th, or the 16th to the 36th nucleotides,from the 5′ end of the 55th exon in the human dystrophin gene isrespectively identical to the nucleotide sequence consisting of the172nd to the 192nd, the 171st to the 192nd, the 170th to the 192nd, the169th to the 192nd, the 168th to the 192nd, the 172nd to the 191st, the171st to the 191st, the 170th to the 191st, the 169th to the 191st, the168th to the 191st, the 172nd to the 190th, the 171st to the 190th, the170th to the 190th, the 169th to the 190th, the 168th to the 190th, the172nd to the 189th, the 171st to the 189th, the 170th to the 189th, the169th to the 189th, the 168th to the 189th, the 172nd to the 188th, the171st to the 188th, the 170th to the 188th, the 169th to the 188th, the168th to the 188th, the 162nd to the 182nd, the 161st to the 182nd, the160th to the 182nd, the 159th to the 182nd, the 158th to the 182nd, the162nd to the 181st, the 161st to the 181st, the 160th to the 181st, the159th to the 181st, the 158th to the 181st, the 162nd to the 180th, the161st to the 180th, the 160th to the 180th, the 159th to the 180th, the158th to the 180th, the 162nd to the 179th, the 161st to the 179th, the160th to the 179th, the 159th to the 179th, the 158th to the 179th, the162nd to the 178th, the 161st to the 178th, the 160th to the 178th, the159th to the 178th, the 158th to the 178th, the 157th to the 179th, the156th to the 179th, the 155th to 179th, the 157th to the 178th, the156th to the 178th, the 155th to the 178th, the 159th to the 177th, the158th to the 177th, the 157th to the 177th, the 156th to the 177th, the155th to the 177th, the 159th to the 176th, the 158th to the 176th, the157th to the 176th, the 156th to the 176th, the 155th to the 176th, the159th to the 175th, the 158th to the 175th, the 157th to the 175th, the156th to the 175th, or the 155th to the 175th nucleotides of SEQ ID NO:5.

The complementary sequence to the nucleotide sequence consisting of the−3rd to the 176th nucleotides, from the 5′ end of exon 45 is representedby SEQ ID NO: 6. Herein, the nucleotide sequence consisting of the −3rdto the −1st nucleotides, from the 5′ end of exon 45 (the nucleotidesequence consisting of the 1st to the 3rd nucleotides of SEQ ID NO: 2)represents the nucleotide sequence consisting of 3 nucleotides at themost 3′ end downstream of intron 44 which is located between exon 44 andexon 45. More specifically, the nucleotide sequence of exon 45 is thenucleotide sequence consisting of the 4th to the 179th nucleotides ofSEQ ID NO: 2 and the complementary sequence to exon 45 is the nucleotidesequence consisting of the 1st to the 176th nucleotides of SEQ ID NO: 6.

Herein, the complementary sequence to the nucleotide sequencesconsisting of the −3rd to the 19th, the −3rd to the 20th, the −3rd tothe 21st, the −3 rd to the 22nd, the −3rd to the 23rd, the −2nd to the19th, the −2nd to the 20th, the −2nd to the 21st, the −2nd to the 22nd,the −2nd to the 23rd, the −1 st to the 19th, the −1st to the 20th, the−1 st to the 21st, the −1st to the 22nd, −1st to the 23rd, the 1st tothe 19th, the 1st to the 20th, the 1st to the 21st, the 1st to the 22nd,the 1st to the 23rd, the 2nd to the 19th, the 2nd to the 20th, the 2ndto the 21st, the 2nd to the 22nd, the 2nd to the 23rd, the −2nd to the24th, the −2nd to the 25th, the −2nd to the 26th, the −2nd to the 27th,the −1st to the 24th, the −1st to the 25th, the −1st to the 26th, the−1st to the 27th, the 1st to the 24th, the 1st to the 25th, the 1st tothe 26th, the 1st to the 27th, the 2nd to the 24th, the 2nd to the 25th,the 2nd to the 26th, the 2nd to the 27th, the 3rd to the 23rd, the 3rdto the 24th, the 3rd to the 25th, the 3rd to the 26th, the 3rd to the27th, the 4th to the 28th, the 4th to the 29th, the 4th to the 30th, the4th to the 31st, the 4th to the 32nd, the 5th to the 28th, the 5th tothe 29th, the 5th to the 30th, the 5th to the 31st, the 5th to the 32nd,the 6th to the 28th, the 6th to the 29th, the 6th to the 30th, the 6thto the 31st, the 6th to the 32nd, the 7th to the 28th, the 7th to the29th, the 7th to the 30th, the 7th to the 31st, the 7th to the 32nd, the8th to the 28th, the 8th to the 29th, the 8th to the 30th, the 8th tothe 31st, or the 8th to the 32nd nucleotides, from the 5′ end of the45th exon in the human dystrophin gene is respectively identical to thenucleotide sequence consisting of the 158th to the 179th, the 157th tothe 179th, the 156th to the 179th, the 155th to the 179th, the 154th tothe 179th, the 158th to the 178th, the 157th to the 178th, the 156th tothe 178th, the 155th to the 178th, the 154th to the 178th, the 158th tothe 177th, the 157th to the 177th, the 156th to the 177th, the 155th tothe 177th, the 154th to the 177th, the 158th to the 176th, the 157th tothe 176th, the 156th to the 176th, the 155th to the 176th, the 154th tothe 176th, the 158th to the 175th, the 157th to the 175th, the 156th tothe 175th, the 155th to the 175th, the 154th to the 175th, the 153rd tothe 178th, the 152nd to the 178th, the 151st to the 178th, the 150th tothe 178th, the 153rd to the 177th, the 152nd to the 177th, the 151st tothe 177th, the 150th to the 177th, the 153rd to the 176th, the 152nd tothe 176th, the 151st to the 176th, the 150th to the 176th, the 153rd tothe 175th, the 152nd to the 175th, the 151st to the 175th, the 150th tothe 175th, the 154th to the 174th, the 153rd to the 174th, the 152nd tothe 174th, the 151st to the 174th, the 150th to the 174th, the 149th tothe 173rd, the 148th to the 173rd, the 147th to the 173rd, the 146th tothe 173rd, the 147th to the 173rd, the 149th to the 172nd, the 148th tothe 172nd, the 147th to the 172nd, the 146th to the 172nd, the 145th tothe 172nd, the 149th to the 171st, the 148th to the 171st, the 147th tothe 171st, the 146th to the 171st, the 145th to the 171st, the 149th tothe 170th, the 148th to the 170th, the 147th to the 170th, the 146th tothe 170th, the 145th to the 170th, the 149th to the 169th, the 148th tothe 169th, the 147th to the 169th, the 146th to the 169th or the 145thto the 169th nucleotides of SEQ ID NO: 6.

The complementary sequence to the nucleotide sequence consisting of the1st to the 109th nucleotides, from the 5′ end of exon 50, and the 1st tothe 20th nucleotides, from the 5′ end of intron 50, is represented bySEQ ID NO: 7. Herein, the nucleotide sequence consisting of the 1st tothe 20th nucleotides, from the 5′ end of intron 50 (the nucleotidesequence consisting of the 110th to the 129th nucleotides of SEQ ID NO:3) is the nucleotide sequence consisting of 20 nucleotides at the most5′ end upstream of intron 50 which is located between exon 50 and exon51. More specifically, the nucleotide sequence of exon 50 is thenucleotide sequence consisting of the 1st to the 109th nucleotides ofSEQ ID NO: 3 and the complementary sequence to exon 50 is the nucleotidesequence consisting of the 21st to the 129th nucleotides of SEQ ID NO:7.

Herein, the complementary sequence to the nucleotide sequencesconsisting of the 105th to the 125th, the 105th to the 126th, the 105thto the 127th, the 105th to the 128th, the 105th to the 129th, the 106thto the 125th, the 106th to the 126th, the 106th to the 127th, the 106thto the 128th, the 106th to the 129th, the 107th to the 125th, the 107thto the 126th, the 107th to the 127th, the 107th to the 128th, the 107thto the 129th, the 108th to the 125th, the 108th to the 126th, the 108thto the 127th, the 108th to the 128th, the 108th to the 129th, the 109thto the 125th, the 109th to the 126th, the 109th to the 127th, the 109thto the 128th or the 109th to the 129th nucleotides, from the 5′ end ofthe 50th exon in the human dystrophin gene is respectively identical tothe nucleotide sequence consisting of the 5th to the 25th, the 4th tothe 25th, the 3rd to the 25th, the 2nd to the 25th, the 1st to the 25th,the 5th to the 24th, the 4th to the 24th, the 3rd to the 24th, the 2ndto the 24th, the 1st to the 24th, the 5th to the 23rd, the 4th to the23rd, the 3rd to the 23rd, the 2nd to the 23rd, the 1st to the 23rd, the5th to the 22nd, the 4th to the 22nd, the 3rd to the 22nd, the 2nd tothe 22nd, the 1st to the 22nd, the 5th to the 21st, the 4th to the 21st,the 3rd to the 21st, the 2nd to the 21st or the 1st to the 21stnucleotides of SEQ ID NO: 7.

The complementary sequence to the nucleotide sequence consisting of the1st to the 148th nucleotides, from the 5′ end of exon 44 is representedby SEQ ID NO: 8.

Herein, the complementary sequence to the nucleotide sequencesconsisting of the 9th to the 30th, the 9th to the 31st, the 9th to the32nd, the 9th to the 33rd, the 9th to the 34th, the 10th to the 30th,the 10th to the 31st, the 10th to the 32nd, the 10th to the 33rd, the10th to the 34th, the 11th to the 30th, the 11th to the 31st, the 11thto the 32nd, the 11th to the 33rd, the 11th to the 34th, the 12th to the30th, the 12th to the 31st, the 12th to the 32nd, the 12th to the 33rd,the 12th to the 34th, the 13th to the 30th, the 13th to the 31st, the13th to the 32nd, the 13th to the 33rd, the 13th to the 34th, the 24thto the 45th, the 24th to the 46th, the 24th to the 47th, the 24th to the48th, the 24th to the 49th, the 25th to the 45th, the 25th to the 46th,the 25th to the 47th, the 25th to the 48th, the 25th to the 49th, the26th to the 45th, the 26th to the 46th, the 26th to the 47th, the 26thto the 48th, the 26th to the 49th, the 27th to the 45th, the 27th to the46th, the 27th to the 47th, the 27th to the 48th, the 27th to the 49th,the 28th to the 45th, the 28th to the 46th, the 28th to the 47th, the28th to the 48th, the 28th to the 49th, the 29th to the 45th, the 29thto the 46th, the 29th to the 47th, the 29th to the 48th or the 29th tothe 49th nucleotides, from the 5′ end of the 44th exon in the humandystrophin gene is respectively identical to the nucleotide sequenceconsisting of the 119th to the 140th, the 118th to the 140th, the 117thto the 140th, the 116th to the 140th, the 115th to the 140th, 119th tothe 139th, the 118th to the 139th, the 117th to the 139th, the 116th tothe 139th, the 115th to the 139th, 119th to the 138th, the 118th to the138th, the 117th to the 138th, the 116th to the 138th, the 115th to the138th, 119th to the 137th, the 118th to the 137th, the 117th to the137th, the 116th to the 137th, the 115th to the 137th, 119th to the136th, the 118th to the 136th, the 117th to the 136th, the 116th to the136th, the 115th to the 136th, the 104th to the 125th, the 103rd to the125th, the 102nd to the 125th, the 101th to the 125th, the 100th to the125th, the 104th to the 124th, the 103rd to the 124th, the 102nd to the124th, the 101st to the 124th, the 100th to the 124th, the 104th to the123rd, the 103rd to the 123rd, the 102nd to the 123rd, the 101st to the123rd, the 100th to the 123rd, the 104th to the 122nd, the 103rd to the122nd, the 102nd to the 122nd, the 101st to the 122nd, the 100th to the122nd, the 104th to the 121st, the 103rd to the 121st, the 102nd to the121st, the 101st to the 121st, the 100th to the 121st, the 104th to the120th, the 103rd to the 120th, the 102nd to the 120th, the 101st to the120th or the 100th to the 120th nucleotides of SEQ ID NO: 8.

The relationship between the location in the nucleotide sequence fromthe 5′ end of the 55th, the 45th, the 50th, and the 44th exon and thelocation in the nucleotide sequence of SEQ ID NO: 5-8 is represented asthe tables below.

TABLE 1 the location of nucleotides the location of corresponding from5′end of exon 55 nucleotides in the nucleotide nucleotide sequencessequences of SEQ ID NO. 5 −2nd~19th 172nd~192nd −2nd~20th 171st~192nd−2nd~21st 170th~192nd −2nd~22nd 169th~192nd −2nd~23rd 168th~192nd−1st~19th 172nd~191st −1st~20th 171st~191st −1st~21st 170th~191st−1st~22nd 169th~191st −1st~23rd 168th~191st 1st~19th 172nd~190th1st~20th 171st~190th 1st~21st 170th~190th 1st~22nd 169th~190th 1st~23rd168th~190th 2nd~19th 172nd~189th 2nd~20th 171st~189th 2nd~21st170th~189th 2nd~22nd 169th~189th 2nd~23rd 168th~189th 3rd~19th172nd~188th 3rd~20th 171st~188th 3rd~21st 170th~188th 3rd~22nd169th~188th 3rd~23rd 168th~188th 9th~29th 162nd~182nd 9th~30th161st~182nd 9th~31st 160th~182nd 9th~32nd 159th~182nd 9th~33rd158th~182nd 10th~29th 162nd~181st 10th~30th 161st~181st 10th~31st160th~181st 10th~32nd 159th~181st 10th~33rd 158th~181st 11th~29th162nd~180th 11th~30th 161st~180th 11th~31st 160th~180th 11th~32nd159th~180th 11th~33rd 158th~180th 12th~29th 162nd~179th 12th~30th161st~179th 12th~31st 160th~179th 12th~32nd 159th~179th 12th~33rd158th~179th 13th~29th 162nd~178th 13th~30th 161st~178th 13th~31st160th~178th 13th~32nd 159th~178th 13th~33rd 158th~178th 12th~34th157th~179th 12th~35th 156th~179th 12th~36th 155th~179th 13th~34th157th~178th 13th~35th 156th~178h 13th~36th 155th~178th 14th~32nd159th~177th 14th~33rd 158th~177th 14th~34th 157th~177th 14th~35th156th~177th 14th~36th 155th~177th 15th~32nd 159th~176th 15th~33rd158th~176th 15th~34th 157th~176th 15th~35th 156th~176th 15th~36th155th~176th 16th~32nd 159th~175th 16th~33rd 158th~175th 16th~34th157th~175th 16th~35th 156th~175th 16th~36th 155th~175th

TABLE 2 the location of nucleotides the location of corresponding from5′end of exon 45 nucleotides in the nucleotide nucleotide sequensessequences in SEQ ID NO. 6 −3rd~19th 158th~179th −3rd~20th 157th~179th−3rd~21st 156th~179th −3rd~22nd 155th~179th −3rd~23rd 154th~179th−2nd~19th 158th~178th −2nd~20th 157th~178th −2nd~21st 156th~178th−2nd~22nd 155th~178th −2nd~23rd 154th~178th −1st~19th 158th~177th−1st~20th 157th~177th −1st~21st 156th~177th −1st~22nd 155th~177th−1st~23rd 154th~177th 1st~19th 158th~176th 1st~20th 157th~176th 1st~21st156th~176th 1st~22nd 155th~176th 1st~23rd 154th~176th 2nd~19th158th~175th 2nd~20th 157th~175th 2nd~21st 156th~175th 2nd~22nd155th~175th 2nd~23rd 154th~175th −2nd~24th 153rd~178th −2nd~25th152nd~178th −2nd~26th 151st~178th −2nd~27th 150th~178th −1st~24th153rd~177th −1st~25th 152nd~177th −1st~26th 151st~177th −1st~27th150th~177th 1st~24th 153rd~176th 1st~25th 152nd~176th 1st~26th151st~176th 1st~27th 150th~176th 2nd~24th 153rd~175th 2nd~25th152nd~175th 2nd~26th 151st~175h 2nd~27th 150th~175th 3rd~23rd154th~174th 3rd~24th 153rd~174th 3rd~25th 152nd~174th 3rd~26th151st~174th 3rd~27th 150th~174th 4th~28th 149th~173rd 4th~29th148th~173rd 4th~30th 147th~173rd 4th~31st 146th~173rd 4th~32nd147th~173rd 5th~28th 149th~172nd 5th~29th 148th~172nd 5th~30th147th~172nd 5th~31st 146th~172nd 5th~32nd 145th~172nd 6th~28th149th~171st 6th~29th 148th~171st 6th~30th 147th~171st 6th~31st146th~171st 6th~32nd 145th~171st 7th~28th 149th~170th 7th~29th148th~170th 7th~30th 147th~170th 7th~31st 146th~170th 7th~32nd145th~170th 8th~28th 149th~169th 8th~29th 148th~169th 8th~30th147th~169th 8th~31st 146th~169th 8th~32nd 145th~169th

TABLE 3 the location of nucleotides the location of corresponding from5′end of exon 50 nucleotides in the nucleotide nucleotide sequencessequences in SEQ ID NO. 7 105th~125th 5th~25th 105th~126th 4th~25th105th~127th 3rd~25th 105th~128th 2nd~25th 105th~129th 1st~25th106th~125th 5th~24th 106th~126th 4th~24h 106th~127th 3rd~24th 106th~128h2nd~24th 106th~129th 1st~24th 107th~125th 5th~23rd 107th~126h 4th~23rd107th~127th 3rd~23rd 107th~128th 2nd~23rd 107th~129th 1st~23rd108th~125th 5th~22nd 108th~126th 4th~22nd 108th~127h 3rd~22nd108th~128th 2nd~22nd 108th~129th 1st~22nd 109th~125th 5th~21st109th~126th 4th~21st 109th~127th 3rd~21st 109th~128th 2nd~21st109th~129h 1st~21st

TABLE 4 the location of nucleotides the location of corresponding from5′end of exon 44 nucleotides in the nucleotide nucleotide sequencessequences in SEQ ID NO. 8 9th~30th 119th~140th 9th~31st 118th~140th9th~32nd 117th~140th 9th~33rd 116th~140th 9th~34th 115th~140th 10th~30th119th~139th 10th~31st 118th~139th 10th~32nd 117th~139th 10th~33rd116th~139th 10th~34th 115th~139th 11th~30th 119th~138th 11th~31st118th~138th 11th~32nd 117th~138th 11th~33rd 116th~138th 11th~34th115th~138th 12th~30th 119th~137th 12th~31st 118th~137th 12th~32nd117th~137th 12th~33rd 116th~137th 12th~34th 115th~137th 13th~30th119th~136th 13th~31st 118th~136th 13th~32nd 117th~136th 13th~33rd116th~136th 13th~34th 115th~136th 24th~45th 104th~125th 24th~46th103rd~125th 24th~47th 102nd~125th 24th~48th 101st~125th 24th~49th100th~125h 25th~45th 104th~124th 25th~46th 103rd~124th 25th~47th102nd~124th 25th~48th 101st~124th 25th~49th 100th~124th 26th~45th104th~123rd 26th~46th 103rd~123rd 26th~47th 102nd~123rd 26th~48th101st~123rd 26th~49th 100th~123rd 27th~45th 104th~122nd 27th~46th103rd~122nd 27th~47th 102nd~122nd 27th~48th 101st~122nd 27th~49th100th~122nd 28th~45th 104th~121st 28th~46th 103rd~121st 28th~47th102nd~121st 28th~48th 101st~121st 28th~49th 100th~121st 29th~45th104th~120th 29th~46th 103rd~120th 29th~47th 102nd~120th 29th~48th101st~120th 29th~49th 100th~120th

It is preferred that the exon 55 skipping oligomer of the presentinvention consists of a complementary sequence to any one of thenucleotide sequences consisting of the 1st to the 21st, the 11th to the31st or the 14th to the 34th nucleotides, from the 5′ end of the 55thexon in the human dystrophin gene (e.g., any one of the sequencesconsisting of the 170th to the 190th, the 160th to the 180th or the157th to the 177th of SEQ ID NO: 5).

It is preferred that the exon 45 skipping oligomer of the presentinvention consists of a complementary sequence to any one of thenucleotide sequences consisting of the −2nd to the 19th, the 1st to the21st, the 1st to the 25th or the 6th to the 30th nucleotides, from the5′ end of the 45th exon in the human dystrophin gene (e.g., any one ofthe sequences consisting of the 158th to the 178th, 156th to the 176th,the 152nd to the 176th or the 147th to the 171st nucleotides of SEQ IDNO: 6).

It is preferred that the exon 50 skipping oligomer of the presentinvention consists of a complementary sequence to any one of thenucleotide sequences consisting of the 106th to the 126th or the 107thto the 127th nucleotides, from the 5′ end of the 50th exon in the humandystrophin gene (e.g., any one of the sequences consisting of the 4th tothe 24th or the 3rd to the 23rd nucleotides of SEQ ID NO: 7).

It is preferred that the exon 44 skipping oligomer of the presentinvention consists of a complementary sequence to any one of thenucleotide sequences consisting of the 11th to the 32nd, the 25th to the45th, the 26th to the 46th, the 26th to the 47th or the 27th to the 47thnucleotides, from the 5′ end of the 44th exon in the human dystrophingene (e.g., any one of the sequences consisting of the 117th to the138th, the 104th to the 124th, the 103rd to the 123rd, the 102nd to the123rd or the 102nd to the 122nd nucleotides of SEQ ID NO: 8).

The term “cause skipping of the 55th exon in the human dystrophin gene”is intended to mean that by binding of the oligomer of the presentinvention to the site corresponding to exon 55 of the transcript (e.g.,pre-mRNA) of the human dystrophin gene, for example, the nucleotidesequence corresponding to the 5′ end of exon 56 is ligated to the 3′side of the nucleotide sequence corresponding to the 3′ end of exon 53in DMD patients with deletion of exon 54 when the transcript is spliced,thus resulting in formation of mature mRNA which is free of codon frameshift.

The term “cause skipping of the 45th exon in the human dystrophin gene”is intended to mean that by binding of the oligomer of the presentinvention to the site corresponding to exon 45 of the transcript (e.g.,pre-mRNA) of the human dystrophin gene, for example, the nucleotidesequence corresponding to the 5′ end of exon 46 is ligated to the 3′side of the nucleotide sequence corresponding to the 3′ end of exon 43in DMD patients with deletion of exon 44 when the transcript is spliced,thus resulting in formation of mature mRNA which is free of codon frameshift.

The term “cause skipping of the 50th exon in the human dystrophin gene”is intended to mean that by binding of the oligomer of the presentinvention to the site corresponding to exon 50 of the transcript (e.g.,pre-mRNA) of the human dystrophin gene, for example, the nucleotidesequence corresponding to the 5′ end of exon 52 is ligated to the 3′side of the nucleotide sequence corresponding to the 3′ end of exon 49in DMD patients with deletion of exon 51 when the transcript is spliced,thus resulting in formation of mature mRNA which is free of codon frameshift.

The term “cause skipping of the 44th exon in the human dystrophin gene”is intended to mean that by binding of the oligomer of the presentinvention to the site corresponding to exon 44 of the transcript (e.g.,pre-mRNA) of the human dystrophin gene, for example, the nucleotidesequence corresponding to the 5′ end of exon 46 is ligated to the 3′side of the nucleotide sequence corresponding to the 3′ end of exon 43in DMD patients with deletion of, exon 45 when the transcript isspliced, thus resulting in formation of mature mRNA which is free ofcodon frame shift.

Accordingly, it is not required for the oligomer of the presentinvention to have a nucleotide sequence 100% complementary to eachtarget sequence, as far as it causes exon 55, 45, 50 or 44 skipping inthe human dystrophin gene. The oligomer of the present invention mayinclude, for example, 1 to 3, 1 or 2, or one nucleotidenon-complementary to the target sequence.

Herein, the term “binding” described above is intended to mean that whenthe oligomer of the present invention is mixed with the transcript ofhuman dystrophin gene, both are hybridized under physiologicalconditions to form a double strand. The term “under physiologicalconditions” refers to conditions set to mimic the in vivo environment interms of pH, salt composition and temperature. The conditions are, forexample, 25 to 40° C., preferably 37° C., pH 5 to 8, preferably pH 7.4and 150 mM of sodium chloride concentration.

Whether the skipping of exon 55, 45, 50 or 44 in the human dystrophingene is caused or not can be confirmed by introducing the oligomer ofthe present invention into a dystrophin expressing cell (e.g., humanrhabdomyosarcoma cells), amplifying the region surrounding exon 55, 45,50 or 44 of mRNA of the human dystrophin gene by RT-PCR from the totalRNA of the dystrophin expressing cell and performing nested PCR orsequence analysis on the PCR amplified product.

The skipping efficiency can be determined as follows. The mRNA for thehuman dystrophin gene is collected from test cells; in the mRNA, thepolynucleotide level “A” of the band where exon 55, 45, 50 or 44 isskipped and the polynucleotide level “B” of the band where exon 55, 45,50 or 44 is not skipped are measured. Using these measurement values of“A” and “B,” the efficiency is calculated by the following equation:

Skipping efficiency (%)=A/(A+B)×100

The oligomer of the present invention includes, for example, anoligonucleotide, morpholino oligomer or peptide nucleic acid (PNA),having a length of 18 to 28 nucleotides. The length is preferably from15 to 30 nucleotides or 20 to 25 nucleotides and morpholino oligomersare preferred.

The oligonucleotide described above (hereinafter referred to as “theoligonucleotide of the present invention”) is the oligomer of thepresent invention composed of nucleotides as constituent units. Suchnucleotides may be any of ribonucleotides, deoxyribonucleotides andmodified nucleotides.

The modified nucleotide refers to one having fully or partly modifiednucleobases, sugar moieties and/or phosphate-binding regions, whichconstitute the ribonucleotide or deoxyribonucleotide.

The nucleobase includes, for example, adenine, guanine, hypoxanthine,cytosine, thymine, uracil, and modified bases thereof. Examples of suchmodified nucleobases include, but not limited to, pseudouracil,3-methyluracil, dihydrouracil, 5-alkylcytosines (e.g.,5-methylcytosine), 5-alkyluracils (e.g., 5-ethyluracil), 5-halouracils(5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidines (6-methyluracil),2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5′-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine,2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine,N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil,5-methylaminomethyluracil, 5-methylcarbonylmethyluracil,5-methyloxyuracil, 5-methyl-2-thiouracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid,2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine,indole, imidazole, xanthine, etc.

Modification of the sugar moiety may include, for example, modificationsat the 2′-position of ribose and modifications of the other positions ofthe sugar. The modification at the 2′-position of ribose includesreplacement of the 2′-OH of ribose with OR, R, R′OR, SH, SR, NH₂, NHR,NR₂, N₃, CN, F, Cl, Br or I, wherein R represents an alkyl or an aryland R′ represents an alkylene.

The modification for the other positions of the sugar includes, forexample, replacement of O at the 4′ position of ribose or deoxyribosewith S, bridging between 2′ and 4′ positions of the sugar, e.g., LNA(Locked Nucleic Acid) or ENA (2′-O,4′-C-Ethylene-bridged Nucleic Acids),but is not limited thereto.

A modification of the phosphate-binding region includes, for example, amodification of replacing phosphodiester bond with phosphorothioatebond, phosphorodithioate bond, alkyl phosphonate bond, phosphoroamidatebond or boranophosphate bond (Enya et al: Bioorganic & MedicinalChemistry, 2008, 18, 9154-9160) (cf., e.g., Japan DomesticRe-Publications of PCT Application Nos. 2006/129594 and 2006/038608).

The alkyl is preferably a straight or branched alkyl having 1 to 6carbon atoms. Specific examples include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec butyl, tert-butyl, n-pentyl,isopentyl, neopentyl, tert-pentyl, n-hexyl and isohexyl. The alkyl mayoptionally be substituted. Examples of such substituents are a halogen,an alkoxy, cyano and nitro. The alkyl may be substituted with 1 to 3substituents.

The cycloalkyl is preferably a cycloalkyl having 5 to 12 carbon atoms.Specific examples include cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclodecyl and cyclododecyl.

The halogen includes fluorine, chlorine, bromine and iodine.

The alkoxy is a straight or branched alkoxy having 1 to 6 carbon atomssuch as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy,sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy,isohexyloxy, etc. Among others, an alkoxy having 1 to 3 carbon atoms ispreferred.

The aryl is preferably an aryl having 6 to 10 carbon atoms. Specificexamples include phenyl, α-naphthyl and β-naphthyl. Among others, phenylis preferred. The aryl may optionally be substituted. Examples of suchsubstituents are an alkyl, a halogen, an alkoxy, cyano and nitro. Thearyl may be substituted with one to three of such substituents.

The alkylene is preferably a straight or branched alkylene having 1 to 6carbon atoms. Specific examples include methylene, ethylene,trimethylene, tetramethylene, pentamethylene, hexamethylene, 2-(ethyl)trimethylene and 1-(methyl) tetramethylene.

The acyl includes a straight or branched alkanoyl or aroyl. Examples ofthe alkanoyl include formyl, acetyl, 2-methylacetyl, 2,2-dimethylacetyl,propionyl, butyryl, isobutyryl, pentanoyl, 2,2-dimethylpropionyl,hexanoyl, etc. Examples of the aroyl include benzoyl, toluoyl andnaphthoyl. The aroyl may optionally be substituted at substitutablepositions and may be substituted with an alkyl(s).

Preferably, the oligonucleotide of the present invention is the oligomerof the present invention containing a constituent unit represented bygeneral formula below wherein the —OH group at position 2′ of ribose issubstituted with methoxy and the phosphate-binding region is aphosphorothioate bond:

wherein Base represents a nucleobase.

The oligonucleotide of the present invention may be easily synthesizedusing various automated synthesizer (e.g., AKTA oligopilot plus 10/100(GE Healthcare)). Alternatively, the synthesis may also be entrusted toa third-party organization (e.g., Promega Inc., or Takara Co.), etc.

The morpholino oligomer of the present invention is the oligomer of thepresent invention comprising the constituent unit represented by generalformula below:

wherein Base has the same significance as defined above, and,W represents a group shown by any one of the following groups:

wherein

-   -   X represents —CH₂R¹, —O—CH₂R¹, —S—CH₂R¹, —NR²R³ or F;    -   R¹ represents H or an alkyl;    -   R² and R³, which may be the same or different, each represents        H, an alkyl, a cycloalkyl or an aryl;    -   Y₁ represents O, S, CH₂ or NR¹;    -   Y₂ represents O, S or NR¹;    -   Z represents O or S.

Preferably, the morpholino oligomer is an oligomer comprising aconstituent unit represented by general formula below(phosphorodiamidate morpholino oligomer (hereinafter referred to as“PMO”)).

wherein Base, R² and R³ have the same significance as defined above.

The morpholino oligomer may be produced in accordance with, e.g., WO1991/009033 or WO 2009/064471. In particular, PMO can be produced by theprocedure described in WO 2009/064471 or produced by the process shownbelow.

[Method for Producing PMO]

An embodiment of PMO is, for example, the compound represented bygeneral formula (I) below (hereinafter PMO

wherein Base, R² and R³ have the same significance as defined above;and, n is a given integer of 1 to 99, preferably a given integer of 18to 28.

PMO (I) can be produced in accordance with a known method, for example,can be produced by performing the procedures in the following steps.

The compounds and reagents used in the steps below are not particularlylimited so long as they are commonly used to prepare PMO.

Also, the following steps can all be carried out by the liquid phasemethod or the solid phase method (using manuals or commerciallyavailable solid phase automated synthesizers). In producing PMO by thesolid phase method, it is desired to use automated synthesizers in viewof simple operation procedures and accurate synthesis.

(1) Step A:

The compound represented by general formula (II) below (hereinafterreferred to as Compound (II)) is reacted with an acid to prepare thecompound represented by general formula (III) below (hereinafterreferred to as Compound (III)):

wherein n, R² and R³ have the same significance as defined above;each B^(P) independently represents a nucleobase which may optionally beprotected;T represents trityl, monomethoxytrityl or dimethoxytrityl; and,L represents hydrogen, an acyl or a group represented by general formula(IV) below (hereinafter referred to as group (IV)).

The “nucleobase” for B^(P) includes the same “nucleobase” as in Base,provided that the amino or hydroxy group in the nucleobase shown byB^(P) may be protected.

Such protective group for amino is not particularly limited so long asit is used as a protective group for nucleic acids. Specific examplesinclude benzoyl, 4-methoxybenzoyl, acetyl, propionyl, butyryl,isobutyryl, phenylacetyl, phenoxyacetyl, 4-tert-butylphenoxyacetyl,4-isopropylphenoxyacetyl and (dimethylamino)methylene. Specific examplesof the protective group for the hydroxy group include 2-cyanoethyl,4-nitrophenethyl, phenylsulfonylethyl, methylsulfonylethyl andtrimethylsilylethyl, and phenyl, which may be substituted by 1 to 5electron-withdrawing group at optional substitutable positions,diphenylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl,methylphenylcarbamoyl, 1-pyrolidinylcarbamoyl, morpholinocarbamoyl,4-(tert-butylcarboxy) benzyl, 4-[(dimethylamino)carboxy]benzyl and4-(phenylcarboxy)benzyl, (cf., e.g., WO 2009/064471).

The “solid carrier” is not particularly limited so long as it is acarrier usable for the solid phase reaction of nucleic acids. It isdesired for the solid carrier to have the following properties: e.g.,(i) it is sparingly soluble in reagents that can be used for thesynthesis of morpholino nucleic acid derivatives (e.g., dichloromethane,acetonitrile, tetrazole, N-methylimidazole, pyridine, acetic anhydride,lutidine, trifluoroacetic acid); (ii) it is chemically stable to thereagents usable for the synthesis of morpholino nucleic acidderivatives; (iii) it can be chemically modified; (iv) it can be chargedwith desired morpholino nucleic acid derivatives; (v) it has a strengthsufficient to withstand high pressure through treatments; and (vi) ithas a uniform particle diameter range and distribution. Specifically,swellable polystyrene (e.g., aminomethyl polystyrene resin 1%dibenzylbenzene crosslinked (200-400 mesh) (2.4-3.0 mmol/g)(manufactured by Tokyo Chemical Industry), Aminomethylated PolystyreneResin [dibenzylbenzene 1%, 100-200 mesh] (manufactured by PeptideInstitute, Inc.)), non-swellable polystyrene (e.g., Primer Support(manufactured by GE Healthcare)), PEG chain-attached polystyrene (e.g.,NH₂-PEG resin (manufactured by Watanabe Chemical Co.), TentaGel resin),controlled pore glass (controlled pore glass; CPG) (manufactured by,e.g., CPG), oxalyl-controlled pore glass (cf., Alul et al., NucleicAcids Research, Vol. 19, 1527 (1991)), TentaGelsupport-aminopolyethylene glycol-derivatized support (cf., e.g., Wrightet al., Tetrahedron Letters, Vol. 34, 3373 (1993)), and a copolymer ofPoros-polystyrene/divinylbenzene.

A “linker” which can be used is a known linker generally used to connectnucleic acids or morpholino nucleic acid derivatives. Examples include3-aminopropyl, succinyl, 2,2′-diethanolsulfonyl and a long chain alkylamino (LCAA).

This step can be performed by reacting Compound (II) with an acid.

The “acid” which can be used in this step includes, for example,trifluoroacetic acid, dichloroacetic acid and trichloroacetic acid. Theacid used is appropriately in a range of, for example, 0.1 molequivalent to 1000 mol equivalents based on 1 mol of Compound (II),preferably in a range of 1 mol equivalent to 100 mol equivalents basedon 1 mol of Compound (II).

An organic amine can be used in combination with the acid describedabove. The organic amine is not particularly limited and includes, forexample, triethylamine. The amount of the organic amine used isappropriately in a range of, e.g., 0.01 mol equivalent to 10 molequivalents, and preferably in a range of 0.1 mol equivalent to 2 molequivalents, based on 1 mol of the acid.

When a salt or mixture of the acid and the organic amine is used in thisstep, the salt or mixture includes, for example, a salt or mixture oftrifluoroacetic acid and triethylamine, and more specifically, a mixtureof 1 equivalent of triethylamine and 2 equivalents of trifluoroaceticacid.

The acid which can be used in this step may also be used in the form ofa dilution with an appropriate solvent in a concentration of 0.1% to30%. The solvent is not particularly limited as far as it is inert tothe reaction, and includes, for example, dichloromethane, acetonitrile,an alcohol (ethanol, isopropanol, trifluoroethanol, etc.), water, or amixture thereof.

The reaction temperature in the reaction described above is preferablyin a range of, e.g., 10° C. to 50° C., more preferably, in a range of20° C. to 40° C., and most preferably, in a range of 25° C. to 35° C.

The reaction time may vary depending upon kind of the acid used andreaction temperature, and is appropriately in a range of 0.1 minute to24 hours in general, and preferably in a range of 1 minute to 5 hours.

After completion of this step, a base may be added, if necessary, toneutralize the acid remained in the system. The “base” is notparticularly limited and includes, for example, diisopropylamine. Thebase may also be used in the form of a dilution with an appropriatesolvent in a concentration of 0.1% (v/v) to 30% (v/v).

The solvent used in this step is not particularly limited so long as itis inert to the reaction, and includes dichloromethane, acetonitrile, analcohol (ethanol, isopropanol, trifluoroethanol, etc.), water, and amixture thereof. The reaction temperature is preferably in a range of,e.g., 10° C. to 50° C., more preferably, in a range of 20° C. to 40° C.,and most preferably; in a range of 25° C. to 35° C.

The reaction time may vary depending upon kind of the base used andreaction temperature, and is appropriately in a range of 0.1 minute to24 hours in general, and preferably in a range of 1 minute to 5 hours.

In Compound (II), the compound of general formula (IIa) below(hereinafter Compound (IIa)), wherein n is 1 and L is a group (IV), canbe produced by the following procedure.

wherein B^(P), T, linker and solid carrier have the same significance asdefined above.

Step 1:

The compound represented by general formula (V) below is reacted with anacylating agent to prepare the compound represented by general formula(VI) below (hereinafter referred to as Compound (VI)).

wherein B^(P), T and linker have the same significance as defined above;and,R⁴ represents hydroxy, a halogen or amino.

This step can be carried out by known procedures for introducinglinkers, using Compound (V) as the starting material.

In particular, the compound represented by general formula (VIa) belowcan be produced by performing the method known as esterification, usingCompound (V) and succinic anhydride.

wherein B^(P) and T have the same significance as defined above.

Step 2:

Compound (VI) is reacted with a solid career by a condensing agent toprepare Compound (IIa).

wherein B^(P), R⁴, T, linker and solid carrier have the samesignificance as defined above.

This step can be performed using Compound (VI) and a solid carrier inaccordance with a process known as condensation reaction.

In Compound (II), the compound represented by general formula (IIa2)below wherein n is 2 to 99 and L is a group represented by generalformula (IV) can be produced by using Compound (IIa) as the startingmaterial and repeating step A and step B of the PMO production methoddescribed in the specification for a desired number of times.

wherein B^(P), R², R³, T, linker and solid carrier have the samesignificance as defined above; and,n′ represents 1 to 98.

In Compound (II), the compound of general formula (IIb) below wherein nis 1 and L is hydrogen can be produced by the procedure described in,e.g., WO 1991/009033.

wherein B^(P) and T have the same significance as defined above.

In Compound (II), the compound represented by general formula (IIb2)below wherein n is 2 to 99 and L is hydrogen can be produced by usingCompound (IIb) as the starting material and repeating step A and step Bof the PMO production method described in the specification for adesired number of times.

wherein B^(P), n′, R², R³ and T have the same significance as definedabove.

In Compound (II), the compound represented by general formula (IIc)below wherein n is 1 and L is an acyl can be produced by performing theprocedure known as acylation reaction, using Compound (Jib).

wherein B^(P) and T have the same significance as defined above; and,R⁵ represents an acyl.

In Compound (II), the compound represented by general formula (IIc2)below wherein n is 2 to 99 and L is an acyl can be produced by usingCompound (IIc) as the starting material and repeating step A and step Bof the PMO production method described in the specification for adesired number of times.

wherein B^(P), n′, R², R³, R⁵ and T have the same significance asdefined above.

(2) Step B

Compound (III) is reacted with a morpholino monomer compound in thepresence of a base to prepare the compound represented by generalformula (VII) below (hereinafter referred to as Compound (VII)):

wherein B^(P), L, n, R², R³ and T have the same significance as definedabove.

This step can be performed by reacting Compound (III) with themorpholino monomer compound in the presence of a base.

The morpholino monomer compound includes, for example, compoundsrepresented by general formula (VIII) below:

wherein B^(P), R², R³ and T have the same significance as defined above.

The “base” which can be used in this step includes, for example,diisopropylamine, triethylamine and N-ethylmorpholine. The amount of thebase used is appropriately in a range of 1 mol equivalent to 1000 molequivalents based on 1 mol of Compound preferably, 10 mol equivalents to100 mol equivalents based on 1 mol of Compound (III).

The morpholino monomer compound and base which can be used in this stepmay also be used as a dilution with an appropriate solvent in aconcentration of 0.1% to 30%. The solvent is not particularly limited asfar as it is inert to the reaction, and includes, for example,N,N-dimethylimidazolidone, N-methylpiperidone, DMF, dichloromethane,acetonitrile, tetrahydrofuran, or a mixture thereof.

The reaction temperature is preferably in a range of, e.g., 0° C. to100° C., and more preferably, in a range of 10° C. to 50° C.

The reaction time may vary depending upon kind of the base used andreaction temperature, and is appropriately in a range of 1 minute to 48hours in general, and preferably in a range of 30 minutes to 24 hours.

Furthermore, after completion of this step, an acylating agent can beadded, if necessary. The “acylating agent” includes, for example, aceticanhydride, acetyl chloride and phenoxyacetic anhydride. The acylatingagent may also be used as a dilution with an appropriate solvent in aconcentration of 0.1% to 30%. The solvent is not particularly limited asfar as it is inert to the reaction, and includes, for example,dichloromethane, acetonitrile, an alcohol(s) (ethanol, isopropanol,trifluoroethanol, etc.), water, or a mixture thereof.

If necessary, a base such as pyridine, lutidine, collidine,triethylamine, diisopropylethylamine, N-ethylmorpholine, etc. may alsobe used in combination with the acylating agent. The amount of theacylating agent is appropriately in a range of 0.1 mol equivalent to10000 mol equivalents, and preferably in a range of 1 mol equivalent to1000 mol equivalents. The amount of the base is appropriately in a rangeof, e.g., 0.1 mol equivalent to 100 mol equivalents, and preferably in arange of 1 mol equivalent to 10 mol equivalents, based on 1 mol of theacylating agent.

The reaction temperature in this reaction is preferably in a range of10° C. to 50° C., more preferably, in a range of 10° C. to 50° C., muchmore preferably, in a range of 20° C. to 40° C., and most preferably, ina range of 25° C. to 35° C. The reaction time may vary depending uponkind of the acylating agent used and reaction temperature, and isappropriately in a range of 0.1 minute to 24 hours in general, andpreferably in a range of 1 minute to 5 hours.

(3) Step C:

In Compound (VII) produced in Step B, the protective group is removedusing a deprotecting agent to prepare the compound represented bygeneral formula (IX).

wherein Base, B^(P), L, n, R², R³ and T have the same significance asdefined above.

This step can be performed by reacting Compound (VII) with adeprotecting agent.

The “deprotecting agent” includes, e.g., conc. ammonia water andmethylamine. The “deprotecting agent” used in this step may also be usedas a dilution with, e.g., water, methanol, ethanol, isopropyl alcohol,acetonitrile, tetrahydrofuran, DMF, N,N-dimethylimidazolidone,N-methylpiperidone, or a mixture of these solvents. Among others,ethanol is preferred. The amount of the deprotecting agent used isappropriately in a range of, e.g., 1 mol equivalent to 100000 molequivalents, and preferably in a range of 10 mol equivalents to 1000 molequivalents, based on 1 mol of Compound (VII).

The reaction temperature is appropriately in a range of 15° C. to 75°C., preferably, in a range of 40° C. to 70° C., and more preferably, ina range of 50° C. to 60° C. The reaction time for deprotection may varydepending upon kind of Compound (VII), reaction temperature, etc., andis appropriately in a range of 10 minutes to 30 hours, preferably 30minutes to 24 hours, and more preferably in a range of 5 hours to 20hours.

(4) Step D:

PMO (I) is produced by reacting Compound (Ix) produced in step C with anacid:

wherein Base, n, R², R³ and T have the same significance as definedabove.

This step can be performed by adding an acid to Compound (IX).

The “acid” which can be used in this step includes, for example,trichloroacetic acid, dichloroacetic acid, acetic acid, phosphoric acid,hydrochloric acid, etc. The acid used is appropriately used to allow thesolution to have a pH range of 0.1 to 4.0, and more preferably, in arange of pH 1.0 to 3.0. The solvent is not particularly limited so longas it is inert to the reaction, and includes, for example, acetonitrile,water, or a mixture of these solvents thereof.

The reaction temperature is appropriately in a range of 10° C. to 50°C., preferably, in a range of 20° C. to 40° C., and more preferably, ina range of 25° C. to 35° C. The reaction time for deprotection may varydepending upon kind of Compound (IX), reaction temperature, etc., and isappropriately in a range of 0.1 minute to 5 hours, preferably 1 minuteto 1 hour, and more preferably in a range of 1 minute to 30 minutes.

PMO (I) can be obtained by subjecting the reaction mixture obtained inthis step to conventional means of separation and purification such asextraction, concentration, neutralization, filtration, centrifugalseparation, recrystallization, reversed phase column chromatography C₈to C₁₈, cation exchange column chromatography, anion exchange columnchromatography, gel filtration column chromatography, high performanceliquid chromatography, dialysis, ultrafiltration, etc., alone or incombination thereof. Thus, the desired PMO (I) can be isolated andpurified (cf., e.g., WO 1991/09033).

In purification of PMO (I) using reversed phase chromatography, e.g., asolution mixture of 20 mM triethylamine/acetate buffer and acetonitrilecan be used as an elution solvent.

In purification of PMO (I) using ion exchange chromatography, e.g., asolution mixture of 1 M saline solution and 10 mM sodium hydroxideaqueous solution can be used as an elution solvent.

A peptide nucleic acid is the oligomer of the present invention having agroup represented by the following general formula as the constituentunit:

wherein Base has the same significance as defined above.

Peptide nucleic acids can be prepared by referring to, e.g., thefollowing literatures.

-   1) P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science, 254,    1497 (1991)-   2) M. Egholm, O. Buchardt, P. E. Nielsen, R. H. Berg, Jacs., 114,    1895 (1992)-   3) K. L. Dueholm, M. Egholm, C. Behrens, L. Christensen, H. F.    Hansen, T. Vulpius, K. H. Petersen, R. H. Berg, P. E. Nielsen, O.    Buchardt, J. Org. Chem., 59, 5767 (1994)-   4) L. Christensen, R. Fitzpatrick, B. Gildea, K. H. Petersen, H. F.    Hansen, T. Koch, M. Egholm, O. Buchardt, P. E. Nielsen, J.    Colin, R. H. Berg, J. Pept. Sci., 1, 175 (1995)-   5) T. Koch, H. F. Hansen, P. Andersen, T. Larsen, H. G. Batz, K.    Otteson, H. Orum, J. Pept. Res., 49, 80 (1997)

In the oligomer of the present invention, the 5′ end may be any ofchemical structures (1) to (3) below, and preferably is (3)-OH.

Hereinafter, the groups shown by (1), (2) and (3) above are referred toas “Group (1),” “Group (2)” and “Group (3),” respectively.

2. Pharmaceutical Composition

The oligomer of the present invention causes exon 55, 45, 50 and 44skipping with a higher efficiency as compared to the prior art antisenseoligomers. It is thus expected that conditions of muscular dystrophy canbe relieved with high efficiency by administering the pharmaceuticalcomposition comprising the oligomer of the present invention to DMDpatients. For example, when the pharmaceutical composition comprisingthe oligomer of the present invention is used, the same therapeuticeffects can be achieved even in a smaller dose than that of theoligomers of the prior art. Accordingly, side effects can be alleviatedand such is economical.

In another embodiment, the present invention provides the pharmaceuticalcomposition for the treatment of muscular dystrophy, comprising as anactive ingredient the oligomer of the present invention, apharmaceutically acceptable salt or hydrate thereof (hereinafterreferred to as “the composition of the present invention”).

Examples of the pharmaceutically acceptable salt of the oligomer of thepresent invention contained in the composition of the present inventionare alkali metal salts such as salts of sodium, potassium and lithium;alkaline earth metal salts such as salts of calcium and magnesium; metalsalts such as salts of aluminum, iron, zinc, copper, nickel, cobalt,etc.; ammonium salts; organic amine salts such as salts of t-octylamine,dibenzylamine, morpholine, glucosamine, phenylglycine alkyl ester,ethylenediamine, N-methylglucamine, guanidine, diethylamine,triethylamine, dicyclohexylamine, N,N′-dibenzylethylenediamine,chloroprocaine, procaine, diethanolamine, N-benzylphenethylamine,piperazine, tetramethylammonium, tris(hydroxymethyl)aminomethane;hydrohalide salts such as salts of hydrofluorates, hydrochlorides,hydrobromides and hydroiodides; inorganic acid salts such as nitrates,perchlorates, sulfates, phosphates, etc.; lower alkane sulfonates suchas methanesulfonates, trifluoromethanesulfonates and ethanesulfonates;arylsulfonates such as benzenesulfonates and p-toluenesulfonates;organic acid salts such as acetates, malates, fumarates, succinates,citrates, tartarates, oxalates, maleates, etc.; and, amino acid saltssuch as salts of glycine, lysine, arginine, ornithine, glutamic acid andaspartic acid. These salts may be produced by known methods.Alternatively, the oligomer of the present invention contained in thecomposition of the present invention may be in the form of a hydratethereof.

Administration route for the composition of the present invention is notparticularly limited so long as it is pharmaceutically acceptable routefor administration, and can be chosen depending upon method oftreatment. In view of easiness in delivery to muscle tissues, preferredare intravenous administration, intraarterial administration,intramuscular administration, subcutaneous administration, oraladministration, tissue administration, transdermal administration, etc.Also, dosage forms which are available for the composition of thepresent invention are not particularly limited, and include, forexample, various injections, oral agents, drips, inhalations, ointments,lotions, etc.

In administration of the oligomer of the present invention to patientswith muscular dystrophy, the composition of the present inventionpreferably contains a carrier to promote delivery of the oligomer tomuscle tissues. Such a carrier is not particularly limited as far as itis pharmaceutically acceptable, and examples include cationic carrierssuch as cationic liposomes, cationic polymers, etc., or carriers usingviral envelope. The cationic liposomes are, for example, liposomescomposed of 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoylglyceroland phospholipids as the essential constituents (hereinafter referred toas “liposome A”), Oligofectamine (registered trademark) (manufactured byInvitrogen Corp.), Lipofectin (registered trademark) (manufactured byInvitrogen Corp.), Lipofectamine (registered trademark) (manufactured byInvitrogen Corp.), Lipofectamine 2000 (registered trademark)(manufactured by Invitrogen Corp.), DMRIE-C (registered trademark)(manufactured by Invitrogen Corp.), GeneSilencer (registered trademark)(manufactured by Gene Therapy Systems), TransMessenger (registeredtrademark) (manufactured by QIAGEN, Inc.), TransIT TKO (registeredtrademark) (manufactured by Minis) and Nucleofector II (Lonza). Amongothers, liposome A is preferred. Examples of cationic polymers are JetSI(registered trademark) (manufactured by Qbiogene, Inc.) and Jet-PEI(registered trademark) (polyethylenimine, manufactured by Qbiogene,Inc.). An example of carriers using viral envelop is GenomeOne(registered trademark) (HVJ-E liposome, manufactured by IshiharaSangyo). Alternatively, the medical devices described in Japanese PatentNo. 2924179 and the cationic carriers described in Japanese DomesticRe-Publication PCT Nos. 2006/129594 and 2008/096690 may be used as well.

A concentration of the oligomer of the present invention contained inthe composition of the present invention may vary depending on kind ofthe carrier, etc., and is appropriately in a range of 0.1 nM to 100 μM,preferably in a range of 1 nM to 10 μM, and more preferably in a rangeof 10 nM to 1 μM. A weight ratio of the oligomer of the presentinvention contained in the composition of the present invention and thecarrier (carrier/oligomer of the present invention) may vary dependingon property of the oligomer, type of the carrier, etc., and isappropriately in a range of 0.1 to 100, preferably in a range of 1 to50, and more preferably in a range of 10 to 20.

In addition to the oligomer of the present invention and the carrierdescribed above, pharmaceutically acceptable additives may also beoptionally formulated in the composition of the present invention.Examples of such additives are emulsification aids (e.g., fatty acidshaving 6 to 22 carbon atoms and their pharmaceutically acceptable salts,albumin and dextran), stabilizers (e.g., cholesterol and phosphatidicacid), isotonizing agents (e.g., sodium chloride, glucose, maltose,lactose, sucrose, trehalose), and pH controlling agents (e.g.,hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodiumhydroxide, potassium hydroxide and triethanolamine). One or more ofthese additives can be used. The content of the additive in thecomposition of the present invention is appropriately 90 wt % or less,preferably 70 wt % or less and more preferably, 50 wt % or less.

The composition of the present invention can be prepared by adding theoligomer of the present invention to a carrier dispersion and adequatelystirring the mixture. Additives may be added at an appropriate stepeither before or after addition of the oligomer of the presentinvention. An aqueous solvent that can be used in adding the oligomer ofthe present invention is not particularly limited as far as it ispharmaceutically acceptable, and examples are injectable water orinjectable distilled water, electrolyte fluid such as physiologicalsaline, etc., and sugar fluid such as glucose fluid, maltose fluid, etc.A person skilled in the art can appropriately choose conditions for pHand temperature for such matter.

The composition of the present invention may be prepared into, e.g., aliquid form and its lyophilized preparation. The lyophilized preparationcan be prepared by lyophilizing the composition of the present inventionin a liquid form in a conventional manner. The lyophilization can beperformed, for example, by appropriately sterilizing the composition ofthe present invention in a liquid form, dispensing an aliquot into avial container, performing preliminary freezing for 2 hours atconditions of about −40 to −20° C., performing a primary drying at 0 to10° C. under reduced pressure, and then performing a secondary drying atabout 15 to 25° C. under reduced pressure. In general, the lyophilizedpreparation of the composition of the present invention can be obtainedby replacing the content of the vial with nitrogen gas and capping.

The lyophilized preparation of the composition of the present inventioncan be used in general upon reconstitution by adding an optionalsuitable solution (reconstitution liquid) and redissolving thepreparation. Such a reconstitution liquid includes injectable water,physiological saline and other infusion fluids. A volume of thereconstitution liquid may vary depending on the intended use, etc., isnot particularly limited, and is suitably 0.5 to 2-fold greater than thevolume prior to lyophilization or no more than 500 mL.

It is desired to control a dose of the composition of the presentinvention to be administered, by taking the following factors intoaccount: the type and dosage form of the oligomer of the presentinvention contained; patients' conditions including age, body weight,etc.; administration route; and the characteristics and extent of thedisease. A daily dose calculated as the amount of the oligomer of thepresent invention is generally in a range of 0.1 mg to 10 g/human, andpreferably 1 mg to 1 g/human. This numerical range may vary occasionallydepending on type of the target disease, administration route and targetmolecule. Therefore, a dose lower than the range may be sufficient insome occasion and conversely, a dose higher than the range may berequired occasionally. The composition can be administered from once toseveral times daily or at intervals from one day to several days.

In still another embodiment of the composition of the present invention,there is provided a pharmaceutical composition comprising a vectorcapable of expressing the oligonucleotide of the present invention andthe carrier described above. Such an expression vector may be a vectorcapable of expressing a plurality of the oligonucleotides of the presentinvention. The composition may be formulated with pharmaceuticallyacceptable additives as in the case with the composition of the presentinvention containing the oligomer of the present invention. Aconcentration of the expression vector contained in the composition mayvary depending upon type of the career, etc., and is appropriately in arange of 0.1 nM to 100 μM, preferably in a range of 1 nM to 10 μM, andmore preferably in a range of 10 nM to 1 μM. A weight ratio of theexpression vector contained in the composition and the carrier(carrier/expression vector) may vary depending on property of theexpression vector, type of the carrier, etc., and is appropriately in arange of 0.1 to 100, preferably in a range of 1 to 50, and morepreferably in a range of 10 to 20. The content of the carrier containedin the composition is the same as in the case with the composition ofthe present invention containing the oligomer of the present invention,and a method for producing the same is also the same as in the case withthe composition of the present invention.

EXAMPLES

Hereinafter, the present invention will be described in more detail withreference to EXAMPLES and TEST EXAMPLES below, but is not deemed to belimited thereto.

Reference Example 14-{[(2S,6R)-6-(4-Benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid Loaded onto Amino Polystyrene Resin Step 1:Production of4-([(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl]methoxy-4-oxobutanoicacid

Under argon atmosphere, 3.44 g ofN-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamideand 1.1 g of 4-dimethylaminopyridine (4-DMAP) were suspended in 50 mL ofdichloromethane, and 0.90 g of succinic anhydride was added to thesuspension, followed by stirring at room temperature for 3 hours. To thereaction mixture was added 10 mL of methanol, and the mixture wasconcentrated under reduced pressure. The residue was extracted usingethyl acetate and 0.5 M aqueous potassium dihydrogenphosphate solution.The resulting organic layer was washed sequentially with 0.5 M aqueouspotassium dihydrogenphosphate solution, water and brine in the ordermentioned. The resulting organic layer was dried over sodium sulfate andconcentrated under reduced pressure to give 4.0 g of the product.

Step 2; Production of4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicacid Loaded onto Amino Polystyrene Resin

After 4.0 g of4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicacid was dissolved in 200 mL of pyridine (dehydrated), 0.73 g of 4-DMAPand 11.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride were added to the solution. Then, 25.0 g of aminopolystyrene resin Primer support 200 amino (manufactured GE HealthcareJapan Co., Ltd., 17-5214-97) and 8.5 mL of triethylamine were added tothe mixture, followed by shaking at room temperature for 4 days. Aftercompletion of the reaction, the resin was taken out by filtration. Theresulting resin was washed sequentially with pyridine, methanol anddichloromethane in the order mentioned, and dried under reducedpressure. To the resulting resin were added 200 mL of tetrahydrofuran(dehydrate), 15 mL of acetic anhydride and 15 mL of 2,6-lutidine, andthe mixture was shaken at room temperature for 2 hours. The resin wastaken out by filtration, washed sequentially with pyridine, methanol anddichloromethane in the order mentioned and dried under reduced pressureto give 26.7 g of the product.

The loading amount of the product was determined from the molar amountof the trityl per g resin by measuring UV absorbance at 409 nm using aknown method. The loading amount of the resin was 192.2 μmol/g.

Conditions of UV Measurement

Apparatus: U-2910 (Hitachi, Ltd.)

Solvent: methanesulfonic acid

Wavelength: 265 nm

Wavelength: 26

Reference Example 24-[[(2S,6R)-6-[6-(2-Cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo-butanoicacid Loaded onto Aminopolystyrene Resin Step 1: Production ofN²-(phenoxyacetyl) guanosine

Guanosine, 100 g, was dried at 80° C. under reduced pressure for 24hours. After 500 mL of pyridine (anhydrous) and 500 mL ofdichloromethane (anhydrous) were added thereto, 401 mL ofchlorotrimethylsilane was dropwise added to the mixture under an argonatmosphere at 0° C., followed by stirring at room temperature for 3hours. The mixture was again ice-cooled and 66.3 g of phenoxyacetylchloride was dropwise added thereto. Under ice cooling, the mixture wasstirred for further 3 hours. To the reaction solution was added 500 mLof methanol, and the mixture was stirred at room temperature overnight.The solvent was then removed by distillation under reduced pressure. Theresidue was added with 500 mL of methanol and concentrated under reducedpressure, the process was performed 3 times. To the residue was added 4L of water, and the mixture was stirred for an hour under ice cooling.The precipitates formed were taken out by filtration, washedsequentially with water and cold methanol and then dried to give 150.2 gof the objective compound (yield 102%) (cf.: Org. Lett. (2004), Vol. 6,No. 15, 2555-2557).

Step 2:N⁹-{[(2R,6S)-6-(hydroxymethyl)-4-morpholin-2-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-2-phenoxyacetamidep-toluenesulfonate

In 480 mL of methanol was suspended 30 g of the compound obtained inStep 1, and 130 mL of 2N hydrochloric acid was added to the suspensionunder ice cooling. Subsequently, 56.8 g of ammonium tetraboratetetrahydrate and 16.2 g of sodium periodate were added to the mixture inthe order mentioned and the mixture was stirred at room temperature for3 hours. The reaction mixture was ice cooled and the insoluble matterswere removed off by filtration, followed by washing with 100 mL ofmethanol. The filtrate and washing liquid were combined and the mixturewas ice cooled. To the mixture was added 11.52 g of 2-picoline borane.After stirring for 20 minutes, 54.6 g of p-toluenesulfonic acidmonohydrate was slowly added to the mixture, followed by stirring at 4°C. overnight. The precipitates formed were taken out by filtration andwashed with 500 mL of cold methanol and dried to give 17.7 g of theobjective compound (yield: 43.3%).

¹H NMR (ht. The precipitates were taken o35 (1H, s), 7.55 (2H, m), 7.35(2H, m), 7.10 (2H, d, J=7.82 Hz), 7.00 (3H, m), 5.95 (1H, dd, J=10.64,2.42 Hz), 4.85 (2H, s), 4.00 (1H, m), 3.90-3.60 (2H, m), 3.50-3.20 (5H,m), 2.90 (1H, m), 2.25 (3H, s)

Step 3: Production ofN⁹-{(2R,6S)-6-hydroxymethyl-4-tritylmorpholin-2-yl}-N²-(phenoxyacetyl)guanine

In dichloromethane (30 mL) was suspended 2.0 g of the compound (2.0 g)obtained by Step 2, and triethylamine (13.9 g) and trityl chloride (18.3g) were added to the suspension under ice cooling. The mixture wasstirred at room temperature for an hour. The reaction mixture was washedwith saturated sodium bicarbonate aqueous solution and then with water.The organic layer was collected, dried over magnesium sulfate andconcentrated under reduced pressure. To the residue was added 0.2 Msodium citrate buffer (pH 3)/methanol (1:4 (v/v), 40 mL), and themixture was stirred. Subsequently, water (40 mL) was added and thesuspension mixture was stirred for an hour under ice cooling. Theprecipitates were taken out by filtration, washed with cold methanol anddried to give 1.84 g of the objective compound (yield: 82.0%).

Step 4: Production ofN⁹-[(2R,6S)-6-{(tert-butyldimethylsilyloxy)methyl}-4-tritylmorpholin-2-yl]-N²-(phenoxyacetyl)guanine

In dichloromethane (300 mL) was dissolved the compound (38.3 g) obtainedby Step 3, and imidazole (4.64 g) and t-butyldimethylsilyl chloride(9.47 g) were added to the solution in this order mentioned under icecooling. The reaction solution was stirred at room temperature for anhour. The reaction solution was washed with 0.2 M sodium citrate buffer(pH 3) and then with brine. The organic layer was collected, dried overmagnesium sulfate and concentrated under reduced pressure to give 44.1 gof the objective compound as a crude product.

Step 5: Production ofN⁹-[(2R,6S)-6-{(tert-butyldimethylsilyloxy)methyl}-4-tritylmorpholin-2-yl]-N²-(phenoxyacetyl)-O⁶-triisopropylbenzenesulfonylguanine

In dichloromethane (300 mL) was dissolved the compound (44.1 g) obtainedby Step 4, and 4-dimethylaminopyridine (0.64 g), triethylamine (29.2 mL)and triisopropylbenzensulfonyl chloride (19.0 g) were added to thesolution under ice cooling. The reaction solution was stirred at roomtemperature for an hour. The reaction solution was washed with 1 Maqueous sodium dihydrogenphosphate solution. The organic layer wascollected, dried over magnesium sulfate and concentrated under reducedpressure to give 60.5 g of the objective compound as a crude product.

Step 6: Production of N⁹-[(2R,6S)-6-{(tert-butyldimethylsilyloxy)methyl}-4-tritylmorpholin-2-yl]-N²-(phenoxyacetyl)-O⁶-(2-cyanoethyl)guanine

In dichloromethane (300 mL) was dissolved the compound (60.5 g) obtainedby Step 5, and N-methylpyrrolidine (54.5 mL) was added to the solutionunder ice cooling. The reaction solution was stirred under ice coolingfor an hour. Then, ethylene cyanohydrin (37.2 g), and 1,8-diazabicyclo[5.4.0] undec-7-ene (11.96 g) were added to the solution, and thesolution was stirred under ice cooling for 2 hours. The reactionsolution was washed with 1 M sodium dihydrogenphosphate solution andthen with water. The organic layer was collected, dried over magnesiumsulfate and concentrated under reduced pressure to give 72.4 g of theobjective compound as a crude product.

Step 7: Production ofN⁹-[(2R,6S)-6-hydroxymethyl-4-tritylmorpholin-2-yl]-N²-(phenoxyacetyl)-O⁶-(2-cyanoethyl)guanine

In dichloromethane (300 mL) was dissolved the compound (72.4 g) obtainedin Step 6, and triethylaminetrihydrofluoride (21.1 g) was added to thesolution. The reaction solution was stirred at room temperature for 17hours. The reaction solution was poured into cold saturatedsodiumbicarbonate aqueous solution to neutralize the reaction solution.Then, the dichloromethane layer was collected, dried over magnesiumsulfate and concentrated under reduced pressure. The residue waspurified by a silica gel column chromatography (PSQ100B (manufactured byFUJI SILYSIA CHEMICAL LTD. The same shall apply hereinafter.)) to give14.3 g of the objective compound (yield from Step 4: 39.2%).

Step 8: Production of4-[[(2S,6S)-6-[6-(2-cyanoethoxy)-2-(2-phenoxyacetyl)amino]purin-9-yl-4-tritylmorpholin-2-yl] methoxy]-4-oxo-butanoic acidLoaded onto Amino Polystyrene Resin

The title compound was produced in a manner similar to REFERENCE EXAMPLE1, except thatN⁹-[(2R,6S)-6-hydroxymethyl-4-tritylmorpholin-2-yl]-N²-(phenoxyacetyl)-O⁶-(2-cyanoethyl)guanine was used in this step, instead ofN-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamideused in Step 1 of REFERENCE EXAMPLE 1.

Reference Example 3 4-{[(2S,6R)-6-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicacid Loaded onto Aminopolystyrene Resin

The title compound was produced in a manner similar to REFERENCE EXAMPLE1, except that1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-5-methylpyrimidine-2,4(1H,3H)-dionewas used in this step, instead ofN-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide used in Step 1 of REFERENCE EXAMPLE 1.

Reference Example 44-{[(2S,6R)-6-(6-benzamidepurine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicacid Loaded onto Aminopolystyrene Resin

The title compound was produced in a manner similar to REFERENCE EXAMPLE1, except thatN-{9-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]purine-6-yl}benzamide was used in this step, instead ofN-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide used in Step 1 of REFERENCE EXAMPLE 1.

Reference Example 51,12-Dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoicacid Loaded onto Aminopolystyrene Resin

The title compound was produced in a manner similar to REFERENCE EXAMPLE1, except that 2-[2-(2-hydroxyethoxy)ethoxy]ethyl4-tritylpiperazine-1-carboxylic acid (the compound described inWO2009/064471) was used in this step, instead ofN-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide used in Step 1 of REFERENCE EXAMPLE 1.

Exon 45

According to the descriptions in EXAMPLES 1 to 8 and REFERENCE EXAMPLE 1below, various types of PMO shown by PMO Nos. 1-6 and 8-10 in TABLE 5were synthesized. The PMO synthesized was dissolved in water forinjection (manufactured by Otsuka Pharmaceutical Factory, Inc.). PMO No.7 was purchased from Gene Tools, LLC.

TABLE 5 PMO No. Sequence name Note SEQ ID NO: 1 H45_-2-19(OH) 5′ end:group (3) 9 2 H45_-1-20(OH) 5′ end: group (3) 10 3 H45_1-21(OH) 5′ end:group (3) 11 4 H45_2-22(OH) 5′ end: group (3) 12 5 H45_3-23(OH) 5′ end:group (3) 13 6 H45_-4-21(OH) Sequence corresponding 14 to SEQ ID NO; 30in Patent Document 4, 5′ end.: group (3) 7 H45_5-34(GT) Sequencecorresponding 15 to SEQ ID NO; 4 in Patent Document 3, 5′ end: group (2)8 H45_1-20(OH) 5′ end: group (3) 16 9 H45_2-21(OH) 5′ end: group (3) 1710 H45_1-21(TEG) 5′ end: group (1) 18

Example 1 PMO No. 1

0.2 g4-{[(2S,6R)-6-(4-benzamide-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl]methoxy}4-oxobutanoic acid supported on an aminopolystyrene resin (ReferenceExample 1) (26 μmol) was filled in a column with a filter tip. Then, thesynthetic cycle shown below was started using an nucleic acidsynthesizing machine (AKTA Oligopilot 10 plus). The desired morpholinomonomer compound was added in each coupling cycle to give the nucleotidesequence of the title compound.

TABLE 6 Step Reagent Volume (mL) Time (min) 1 deblocking solution 18-321.8-3.2 2 neutralizing and washing solution 30 1.5 3 coupling solution B5 0.5 4 coupling solution A 1.3  0.25 5 mixture of step 3 and step 4 6.3120-300 reagents mixture 6 acetonitrile 20 1.0 7 capping solution 9 2.08 acetonitrile 30 2.0

The deblocking solution used was dichloromethane containing 3% (w/v)trifluoroacetic acid. The neutralizing and washing solution used was asolution obtained by dissolving N,N-diisopropylethylamine to be 10%(v/v) and tetrahydrofuran to be 5% (v/v) in dichloromethane containing35% (v/v) acetonitrile. The coupling solution A used was a solutionobtained by dissolving the morpholino monomer compound intetrahydrofuran to be 0.10 M. The coupling solution B used was asolution obtained by dissolving N,N-diisopropylethylamine to be 20%(v/v) and tetrahydrofuran to be 1.0% (v/v) in acetonitrile. The cappingsolution used was a solution obtained by dissolving 20% (v/v) aceticanhydride and 30% (v/v) 2,6-lutidine in acetonitrile.

The aminopolystyrene resin loaded with the PMO synthesized above wasrecovered from the reaction vessel and dried at room temperature for atleast 2 hours under reduced pressure. The dried PMO loaded ontoaminopolystyrene resin was charged in a reaction vessel, and 5 mL of 28%ammonia water-ethanol (1/4) was added thereto. The mixture was stirredat 55° C. for 15 hours. The aminopolystyrene resin was separated byfiltration and washed with 1 mL of water-ethanol (1/4). The resultingfiltrate was concentrated under reduced pressure. The resulting residuewas dissolved in 10 mL of a solvent mixture of 20 mM of aceticacid-triethylamine buffer (TEAA buffer) and 10 ml of acetonitrile (4/1)and filtered through a membrane filter. The filtrate obtained waspurified by reversed phase HPLC. The conditions used are as follows.

TABLE 7 Column XBridge 5 μm C18 (Waters, φ 19 × 50 mm, 1 CV = 14 mL)Flow rate 10 mL/min Column temperature room temperature Solution A 20 mMTEAA buffer Solution B CH₃CN Gradient (B) conc. 10→70%/15 CV

Each fraction was analyzed, and the objective product was recovered andconcentrated under reduced pressure. To the concentrated residue wasadded 0.5 mL of 2 M phosphoric acid aqueous solution, and the mixturewas stirred for 1.5 minutes. Furthermore, 2 mL of 2 M sodium hydroxideaqueous solution was added to make the mixture alkaline, followed byfiltration through a membrane filter (0.45 μm).

The resulting aqueous solution containing the objective product waspurified by an anionic exchange resin column. The conditions used are asfollows.

TABLE 8 Column Source 15Q (GE Healthcare, φ 10 × 108 mm, 1 CV = 8.5 mL)Flow rate 8.5 mL/min Column temperature room temperature Solution A 10mM sodium hydroxide aqueous solution Solution B 10 mM sodium hydroxideaqueous solution, 1M sodium chloride aqueous solution Gradient (B) conc.1→50%/40 CV

Each fraction was analyzed (on HPLC) and the objective product wasobtained as an aqueous solution. To the resulting aqueous solution wasadded 0.1 M phosphate buffer (pH 6.0) for neutralization. Next, themixture obtained was demineralized by reversed phase HPLC under theconditions described below.

TABLE 9 Column XBridge 5 μm C8 (Waters, φ 10 × 50 mm, 1 CV = 4 mL) Flowrate 4 mL/min Column temperature 60° C. Solution A water Solution BCH₃CN Gradient (B) conc. 0→50%/20 CV

The objective product was recovered and the mixture was concentratedunder reduced pressure. The resulting residue was dissolved in water.The aqueous solution obtained was freeze-dried to give 1.5 mg of theobjective compound as a white cotton-like solid.

ESI-TOF-MS Clcd.: 6877.8. Found: 6877.4.

Example 2 PMO. No. 3

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

ESI-TOF-MS Clcd.: 6862.8. Found: 6862.5.

Example 3 PMO. No. 2

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

ESI-TOF-MS Clcd.: 6862.8. Found: 6862.3.

Example 4 PMO. No. 4

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purin-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo-butanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 2) was usedas the starting material.

ESI-TOF-MS Clcd.: 6902.8. Found: 6902.3.

Example 5 PMO. No. 5

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 3) was usedas the starting material.

ESI-TOF-MS Clcd.: 6902.8. Found: 6902.4.

Example 6 PMO. No. 8

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

ESI-TOF-MS Clcd.: 6547.5. Found: 6547.2.

Example 7 PMO. No. 9

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

ESI-TOF-MS Clcd.: 6547.5. Found: 6547.2.

Example 8 PMO. No. 10

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that1,12-Dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 5) was usedas the starting material.

ESI-TOF-MS Clcd.: 7214.1. Found: 7213.7.

Comparative Example 1 PMO. No. 6

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

ESI-TOF-MS Clcd.: 8193.9. Found: 8195.3.

Test Example 1 In Vitro Assay

Experiments were performed using the antisense oligomers of2′-O-methoxy-phosphorothioates (2′-OMe-S-RNA) shown by SEQ ID NO: 19 toSEQ ID NO: 35. Various antisense oligomers used for the assay werepurchased from Japan Bio Services. The sequences of various antisenseoligomers are given below.

TABLE 10 Antisense SEQ ID oligomer Nucleotide sequence NO: H45_1-25GCUGCCCAAUGCCAUCCUGGAGUUC 19 H45_6-30 UUGCCGCUGCCCAAUGCCAUCCUGG 20H45_11-35 ACAGUUUGCCGCUGCCCAAUGCCAU 21 H45_16-40UGACAACAGUUUGCCGCUGCCCAAU 22 H45_21-45 UGUUCUGACAACAGUUUGCCGCUGC 23H45_26-50 UUCAAUGUUCUGACAACAGUUUGCC 24 H45_31-55UUGCAUUCAAUGUUCUGACAACAGU 25 H45_36-60 CCCAGUUGCAUUCAAUGUUCUGACA 26H45_41-65 UCUUCCCCAGUUGCAUUCAAUGUUC 27 H45_46-70UUAUUUCUUCCCCAGUUGCAUUCAA 28 H45_51-75 CUGAAUUAUUUCUUCCCCAGUUGCA 29H45_56-80 GAUUGCUGAAUUAUUUCUUCCCCAG 30 H45_61-85UUGAGGAUUGCUGAAUUAUUUCUUC 31 H45_66-90 UGUUUUUGAGGAUUGCUGAAUUAUU 32H45_71-95 GCAUCUGUUUUUGAGGAUUGCUGAA 33 H45_76-100UACUGGCAUCUGUUUUUGAGGAUUG 34 H45_7-31 UUUGCCGCUGCCCAAUGCCAUCCUG 35

RD cells (human rhabdomyosarcoma cell line) were plated at 1×10⁵ in a12-well plate and cultured in 1 mL of Eagle's minimal essential medium(EMEM) (manufactured by Sigma, Inc., hereinafter the same) containing10% fetal calf serum (FCS) (manufactured by Invitrogen Corp.) underconditions of 37° C. and 5% CO₂ overnight. Complexes of variousantisense oligomers (Japan Bio Services) (0.3 or 1 μM) for exon 45skipping and Lipofectamine 2000 (manufactured by Invitrogen Corp.) wereprepared and 100 μL of the complex was added to RD cells where 0.9 mL ofthe medium was exchanged, to reach the final concentration of 30 or 100nM.

After completion of the addition, the cells were cultured overnight. Thecells were washed twice with PBS (manufactured by Nissui, hereafter thesame) and then 250 μL of ISOGEN (manufactured by Nippon Gene) was addedto the cells. After the cells were allowed to stand at room temperaturefor a few minutes for cell lysis, the lysate was collected in anEppendorf tube. The total RNA was extracted according to the protocolattached to ISOGEN. The concentration of the total RNA extracted wasdetermined using a NanoDrop ND-1000 (manufactured by LMS).

RT-PCR was performed with 400 ng of the extracted total RNA using aQIAGEN OneStep RT-PCR Kit. A reaction solution was prepared inaccordance with the protocol attached to the kit. A PTC-100(manufactured by MJ Research) was used as a thermal cycler. The RT-PCRprogram used is as follows.

-   -   50° C., 30 mins: reverse transcription    -   94° C., 15 mins: thermal denaturation    -   [94° C., 30 seconds; 60° C., 30 seconds; 72° C., 1 min]×35        cycles: PCR amplification 72° C., 10 mins:

The nucleotide sequences of the forward primer and reverse primer usedfor RT-PCR are given below.

Forward primer: (SEQ ID NO: 36) 5′-GCTCAGGTCGGATTGACATT-3′ Reverseprimer: (SEQ ID NO: 37) 5′-GGGCAACTCTTCCACCAGTA-3′

The reaction product, 1 μL of the PCR above was analyzed using aBioanalyzer (manufactured by Agilent Technologies, Inc.). Thepolynucleotide level “A” of the band with exon 45 skipping and thepolynucleotide level “B” of the band without exon 45 skipping weremeasured. Based on these measurement values of “A” and “B”, the skippingefficiency was determined by the following equation:

Skipping efficiency (%)=A/(A+B)×100

Experimental Results

The results are shown in FIGS. 1 and 2. These experiments revealed that,when the antisense oligomers were designed at the 1st to the 25th, orthe 6th to the 30th nucleotides from the 5′ end of exon 45 in the humandystrophin gene, exon 45 skipping could be caused with a higherefficiency than that of the antisense oligomer which is designed at the7th to the 31st nucleotides from the 5′ end of exon 45.

Test Example 2 In Vitro Assay

Using an Amaxa Cell Line Nucleofector Kit L on Nucleofector II (Lonza),1, 3, or 10 μM of the oligomers PMO Nos. 1 to 5 and 8 to 10 of thepresent invention and the antisense oligomers PMO Nos. 6 and 7 weretransfected with 3.5×10⁵ of RD cells (human rhabdomyosarcoma cell line).The Program T-030 was used.

After transfection, the cells were cultured for 3 days in 2 mL ofEagle's minimal essential medium (EMEM) (manufactured by Sigma,hereinafter the same) containing 1.0% fetal calf serum (FCS)(manufactured by Invitrogen) under conditions of 37° C. and 5% CO₂. Thecells were washed twice with PBS (manufactured by Nissui, hereinafterthe same) and 500 μL of ISOGEN (manufactured by Nippon Gene) was addedto the cells. After the cells were allowed to stand at room temperaturefor a few minutes to lyse the cells, the lysate was collected in anEppendorf tube. The total RNA was extracted according to the protocolattached to ISOGEN. The concentration of the total RNA extracted wasdetermined using a NanoDrop ND-1000 (manufactured by LMS).

RT-PCR was performed with 400 ng of the extracted total RNA using aQIAGEN OneStep RT-PCR Kit (manufactured by QIAGEN). A reaction solutionwas prepared in accordance with the protocol attached to the kit. APTC-100 (manufactured by MJ Research) was used as a thermal cycler. TheRT-PCR program used is as follows.

-   -   50° C., 30 mins: reverse transcription    -   95° C., 15 mins: thermal denaturation    -   [94° C., 30 seconds; 60° C., 30 seconds; 72° C., 1 min]×35        cycles: PCR amplification 72° C., 10 mins:

The nucleotide sequences of the forward primer and reverse primer usedfor RT-PCR are given below.

Forward primer: (SEQ ID NO: 36) 5′-GCTCAGGTCGGATTGACATT-3′ Reverseprimer: (SEQ ID NO: 37) 5′-GGGCAACTCTTCCACCAGTA-3′The reaction product, 1 μL, of the PCR above was analyzed using aBioanalyzer (manufactured by Agilent Technologies, Inc.).

The polynucleotide level “A” of the band with exon 45 skipping and thepolynucleotide level “B” of the band without exon 45 skipping weremeasured. Based on these measurement values of “A” and “B”, the skippingefficiency was determined by the following equation:

Skipping efficiency (%)=A/(A+B)×100

Experimental Results

The results are shown in FIGS. 3, 4, 14 and 15. These experimentsrevealed that the oligomers PMO Nos. 1 and 3 of the present inventioncaused exon 45 skipping with a equivalent efficiency to the antisenseoligomer PMO No. 6 in RD cells (FIG. 3, 4). In addition, the experimentsrevealed that the oligomers PMO Nos. 1, 2 and 3 of the present inventioncaused exon 45 skipping with a higher efficiency than the antisenseoligomer PMO No. 7 (FIG. 14). Furthermore, the experiments revealed thatthe oligomer PMO No. 3 caused exon 45 skipping with a higher efficiencythan the antisense oligomer PMO No. 10 whose end structure is differentfrom that of PMO No. 3 (FIG. 15).

Test Example 3 In Vitro Assay Using Human Fibroblasts

Human myoD gene (SEQ ID NO: 38) was introduced into the GM05017 cells(human DMD-patient derived fibroblasts, Coriell Institute for MedicalResearch) using a ZsGreen1 coexpression retroviral vector.

After incubation for 4 to 5 days, ZsGreen-positive MyoD-transformedfibroblasts were collected by FACS and plated at 5×10⁴/cm² into a12-well plate. As a growth medium, there was used 1 mL of Dulbecco'sModified Eagle Medium:Nutrient Mixture F-12 (DMEM ⋅ F-12) (InvitrogenCorp.) containing 10% FCS and 1% Penicillin/Streptomycin (P/S)(Sigma-Aldrich, Inc).

The medium was replaced 24 hours later by a differentiation medium(DMEM/F-12 containing 2% equine serum (Invitrogen Corp.), 1% P/S and ITSLiquid Media Supplement (Sigma, Inc.)). The medium was exchanged every 2to 3 days and incubation was continued for 12 to 14 days todifferentiate into myotubes.

Subsequently, the differentiation medium was replaced by adifferentiation medium containing 6 μM Endo-Porter (Gene Tools), and amorpholino oligomer was added thereto at a final concentration of 10 μM.After incubation for 48 hours, total RNA was extracted from the cellsusing a TRIzol (manufactured by Invitrogen Corp.). RT-PCR was performedwith 50 ng of the extracted total RNA using a QIAGEN OneStep RT-PCR Kit.A reaction solution was prepared in accordance with the protocolattached to the kit. An iCycler (manufactured by Bio-Rad) was used as athermal cycler. The RT-PCR program used is as follows.

-   -   50° C., 30 mins: reverse transcription    -   95° C., 15 mins: thermal denaturation    -   [94° C., 1 mins; 60° C., 1 mins; 72° C., 1 mins]×35 cycles: PCR        amplification 72° C., 7 mins: thermal inactivation of polymerase

The primers used were hDMD44F and hDMD46R.

hDMD44F: (SEQ ID NO: 39) 5′-CCTGAGAATTGGGAACATGC-3′ hDMD46R: (SEQ ID NO:40) 5′-TTGCTGCTCTTTTCCAGGTT-3′

The reaction product of RT-PCR above was separated by 2% agarose gelelectrophoresis and gel images were captured with a GeneFlash (Syngene).The polynucleotide level “A” of the band with exon 45 skipping and thepolynucleotide level “B” of the band without exon 45 skipping weremeasured using an Image J (manufactured by National Institutes ofHealth). Based on these measurement values of “A” and “B,” the skippingefficiency was determined by the following equation.

Skipping efficiency (%)=A/(A+B)×100

Experimental Results

The results are shown in FIG. 5. This experiment revealed that theoligomer PMO Nos. 3 of the present invention caused exon 45 skippingwith a high efficiency in GM05017 cells.

Exon 55

According to the descriptions in EXAMPLES 9 to 19 below, various typesof PMO shown by PMO Nos. 11-1.4 and 16-22 in TABLE 11 were synthesized.The PMO synthesized was dissolved in water for injection (manufacturedby Otsuka Pharmaceutical Factory, Inc.). PMO No. 15 was purchased fromGene Tools, LLC.

TABLE 11 PMO No. Sequence name Note SEQ ID NO: 11 H55_2-22(OH) 5′ end:group (3) 41 12 H55_8-28(OH) 5′ end: group (3) 42 13 H55_11-31(OH) 5′end: group (3) 43 14 H55_14-34(OH) 5′ end: group (3) 44 15H55_139-156(GT) Sequence corresponding 115 to h55AON6 in Patent Document5, 5′ end: group (2) 16 H55_12-32(OH) 5′ end: group (3) 45 17H55_13-33(OH) 5′ end: group (3) 46 18 H55_15-35(OH) 5′ end: group (3) 4719 H55_16-36(OH) 5′ end: group (3) 48 20 H55_14-33(OH) 5′ end: group (3)116 21 H55_15-34(OH) 5′ end: group (3) 117 22 H55_14-34(TEG) 5′ end:group (3) 118

Example 9 PMO. No. 11

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that 4-{[(2S,6R)-6-(6-benzamideprine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid loadedonto aminopolystyrene resin (REFERENCE EXAMPLE 4) was used as thestarting material.

ESI-TOF-MS Clcd.: 6807.8. Found: 6807.0.

Example 10 PMO. No. 12

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropirimidine-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene resin(REFERENCE EXAMPLE 3) was used as the starting material.

ESI-TOF-MS Clcd.: 6822.8. Found: 6822.5.

Example 11 PMO. No. 13

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene resin(REFERENCE EXAMPLE 3) was used as the starting material.

ESI-TOF-MS Clcd.: 6837.8. Found: 6837.3.

Example 12 PMO. No. 14

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that 4-[[(2S,6R)-6-(6-benzamideprine-9-yl)-4-tritylmorpholin-2-yl]methoxy]-4-oxobutanoic acid loadedonto aminopolystyrene resin (REFERENCE EXAMPLE 4) was used as thestarting material.

ESI-TOF-MS Clcd.: 6861.8. Found: 6861.4.

Example 13 PMO. No. 16

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 3) was usedas the starting material.

ESI-TOF-MS Clcd.: 6812.8. Found: 6812.7.

Example 14 PMO. No. 17

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo-butanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 2) was usedas the starting material.

ESI-TOF-MS Clcd.: 6852.8. Found: 6852.7.

Example 15 PMO. No. 18

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-[[(2S,6R)-6-[6-(2-cyanoethodxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo-butanoic acidloaded onto aminopolystyrene resin (REFERENCE EXAMPLE 2) was used as thestarting material.

ESI-TOF-MS Clcd.: 6901.8. Found: 6901.5.

Example 16 PMO. No. 19

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 3) was usedas the starting material.

ESI-TOF-MS Clcd.: 6901.8. Found: 6901.7.

Example 17 PMO. No. 20

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-[[(2S,6R)-6-[6-(2-Cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo-butanoic acid loadedonto aminopolystyrene resin (REFERENCE EXAMPLE 2) was used as thestarting material.

ESI-TOF-MS Clcd.: 6522.5. Found: 6522.0.

Example 18 PMO. No. 21

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that 4-{[(2S,6R)-6-(6-benzamideprine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid loadedonto aminopolystyrene resin (REFERENCE EXAMPLE 4) was used as thestarting material.

ESI-TOF-MS Clcd.: 6546.5. Found: 6546.0.

Example 19 PMO. No. 22

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that1,12-dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoicacid (REFERENCE EXAMPLE 5) loaded onto aminopolystyrene resin was usedas the starting material.

ESI-TOF-MS Clcd.: 7213.1. Found: 7212.5.

Test Example 4 In Vitro Assay

Experiments were performed using the antisense oligomers of2′-O-methoxy-phosphorothioates (2′-OMe-S-RNA) shown by SEQ ID NO: 49 toSEQ ID NO: 68. Various antisense oligomers used for the assay werepurchased from Japan Bio Services. The sequences of various antisenseoligomers are given below.

TABLE 12 Antisense SEQ ID oligomer Nucleotide sequence NO: H55_1-21GCAGCCUCUCGCUCACUCACC 49 H55_6-26 CCAAAGCAGCCUCUCGCUCAC 50 H55_11-31UUCUUCCAAAGCAGCCUCUCG 51 H55_21-41 AUCUAUGAGUUUCUUCCAAAG 52 H55_31-51UGUUGCAGUAAUCUAUGAGUU 53 H55_41-61 CAGGGGGAACUGUUGCAGUAA 54 H55_51-71UUUCCAGGUCCAGGGGGAACU 55 H55_61-81 GCAAGAAACUUUUCCAGGUCC 56 H55_71-91UGUAAGCCAGGCAAGAAACUU 57 H55_81-101 UUUCAGCUUCUGUAAGCCAGG 58 H55_91-111UUGGCAGUUGUUUCAGCUUCU 59 H55_101-121 CUGUAGGACAUUGGCAGUUGU 60H55_111-131 GGGUAGCAUCCUGUAGGACAU 61 H55_121-141 CUUUCCUUACGGGUAGCAUCC62 H55_131-151 UUCUAGGAGCCUUUCCUUACG 63 H55_141-161CCUUGGAGUCUUCUAGGAGCC 64 H55_151-171 UCUUUUACUCCCUUGGAGUCU 65H55_161-181 UUUCAUCAGCUCUUUUACUCC 66 H55_171-190 UUGCCAUUGUUUCAUCAGCU 67H55_104-123 UCCUGUAGGACAUUGGCAGU 68

Experiments were performed in accordance with the condition and theprocedure of Exon 45 (TEST EXAMPLE 1), except that the RT-PCR wasperformed using the primers below.

Forward primer: (SEQ ID NO: 69) 5′-CATGGAAGGAGGGTCCCTAT-3′ Reverseprimer: (SEQ ID NO: 70) 5′-CTGCCGGCTTAATTCATCAT-3′

Experimental Results

The results are shown in FIGS. 6 and 7. These experiments revealed that,when the antisense oligomers were designed at the 1st to the 21st, orthe 11th to the 31st nucleotides from the 5′ end of exon 55 in the humandystrophin gene, exon 55 skipping of these antisense oligomers could becaused with a higher efficiency than that of the antisense oligomerwhich is designed at the 104th to the 123rd nucleotides from the 5′ endof exon 55.

Test Example 5 In Vitro Assay

Experiments were performed in accordance with the condition and theprocedure of exon 45 (TEST EXAMPLE 2), except that the RT-PCR wasperformed using the primers below.

Forward primer: (SEQ ID NO: 69) 5′-CATGGAAGGAGGGTCCCTAT-3′ Reverseprimer: (SEQ ID NO: 70) 5′-CTGCCGGCTTAATTCATCAT-3′

Experimental Results

The results are shown in FIGS. 8, 16 and 17. These experiments revealedthat in RD cells, the oligomers PMO Nos. 12, 13 and 14 (H55_8-28 (OH),H55_11-31 (OH) and H55_14-34 (OH)) of the present invention all causedexon 55 skipping with a high efficiency (FIG. 8). Also, the oligomersPMO Nos. 14, 16, 17, 18 and 19 (H55_14-34 (OH), H55_12-32 (OH),H55_13-33 (OH), H55_15-35 (OH) and H55_16-36 (OH)) of the presentinvention were found to cause exon 55 skipping with a notably higherefficiency than that of the antisense oligomer PMO No. 15 (H55_139-156(GT)) in RD cells (FIG. 16). The oligomer PMO No. 14 of the presentinvention and the oligomer PMO No. 21 (H55_15-34(OH)), which is one baseshorter than the oligomer PMO No. 14, were found to cause exon 55skipping with the same efficiency (FIG. 17). Furthermore, theexperiments revealed that the oligomer PMO No. 14 of the presentinvention caused exon 55 skipping with the same efficiency as theoligomer PMO No. 22 (H55_14-34 (TEG)), which has a different endstructure from that of the oligomer PMO No. 14 (FIG. 17).

Exon 44

According to the descriptions in EXAMPLES 20 to 29 below, various typesof PMO shown by PMO Nos. 23-29 and 31-33 in TABLE below weresynthesized. The PMO synthesized was dissolved in water for injection(manufactured by Otsuka Pharmaceutical Factory, Inc.). PMO No. 30 waspurchased from Gene Tools, LLC.

TABLE 13 PMO No. Sequence name Note SEQ ID NO: 23 H44_23-43(OH) 5′ end:group (3) 71 24 H44_25-45(OH) 5′ end: group (3) 72 25 H44_26-46(OH) 5′end: group (3) 73 26 H44_27-47(OH) 5′ end: group (3) 74 27 H44_28-48(OH)5′ end: group (3) 75 28 H44_29-49(OH) 5′ end: group (3) 76 29H44_30-50(OH) 5′ end: group (3) 77 30 H44_10-39(GT) Sequencecorresponding 78 to SEQ ID NO: 1 in Patent Document 3, 5′ end: group (2)31 H44_27-46(OH) 5′ end: group (3) 79 32 H44_28-47(OH) 5′ end: group (3)80 33 H44_27-47(TEG) 5′ end: group (1) 81

Example 20 PMO. No. 23

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 3) was usedas the starting material.

ESI-TOF-MS Clcd.: 6918.9. Found: 6918.3.

Example 21 PMO. No. 24

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene resin(REFERENCE EXAMPLE 3) was used as the starting material.

ESI-TOF-MS Clcd.: 6903.9. Found: 6904.2.

Example 22 PMO. No. 25

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that 4-{[(2S,6R)-6-(6-benzamideprine-9-yl)-4-tritylmorpholin-2-yl}methoxy]-4-oxobutanoic acid loadedonto aminopolystyrene resin (REFERENCE EXAMPLE 4) was used as thestarting material.

ESI-TOF-MS Clcd.: 6912.9. Found: 6912.4.

Example 23 PMO. No. 26

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene resin(REFERENCE EXAMPLE 3) was used as the starting material.

ESI-TOF-MS Clcd.: 6903.9. Found: 6904.2.

Example 24 PMO. No. 27

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that 4-{[(2S,6R)-6-(6-benzamideprine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid loadedonto aminopolystyrene resin (REFERENCE EXAMPLE 4) was used as thestarting material.

ESI-TOF-MS Clcd.: 6927.9. Found: 6927.4.

Example 25 PMO. No. 28

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene resin(REFERENCE EXAMPLE 3) was used as the starting material.

ESI-TOF-MS Clcd.: 6942.9. Found: 6942.3.

Example 26 PMO. No. 29

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that 4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene resin(REFERENCE EXAMPLE 3) was used as the starting material.

ESI-TOF-MS Clcd.: 6917.9. Found: 6918.3.

Example 27 PMO. No. 31

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that 4-{[(2S,6R)-6-(6-benzamideprine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4 oxobutanoic acid loadedonto aminopolystyrene resin (REFERENCE EXAMPLE 4) was used as thestarting material.

ESI-TOF-MS Clcd.: 6573.6. Found: 6572.4.

Example 28 PMO. No. 32

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene resin(REFERENCE EXAMPLE 3) was used as the starting material.

ESI-TOF-MS Clcd.: 6588.6. Found: 6588.3.

Example 29 PMO. No. 33

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that1,12-dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 5) was usedas the starting material.

ESI-TOF-MS Clcd.: 7255.2. Found: 7254.7.

Test Example 6 In Vitro Assay

Experiments were performed using the antisense oligomers of2′-O-methoxy-phosphorothioates (2′-OMe-S-RNA) shown by SEQ ID NO: 82 toSEQ ID NO: 95 and SEQ ID NO: 109 to SEQ ID NO: 118. Various antisenseoligomers used for the assay were purchased from Japan Bio Services. Thesequences of various antisense oligomers are given below.

TABLE 14 Antisense SEQ ID oligomer Nucleotide sequence NO: H44_1-22CUCAACAGAUCUGUCAAAUCGC 82 H44_6-27 CAUUUCUCAACAGAUCUGUCAA 105 H44_11-32GCCGCCAUUUCUCAACAGAUCU 83 H44_16-37 AAAACGCCGCCAUUUCUCAACA 106 H44_21-42UAAUGAAAACGCCGCCAUUUCU 84 H44_26-47 UAUCAUAAUGAAAACGCCGCCA 85 H44_31-52CUUUAUAUCAUAAUGAAAACGC 86 H44_36-57 AAUAUCUUUAUAUCAUAAUGAA 107 H44_41-62GAUUAAAUAUCUUUAUAUCAUA 87 H44_51-72 GUUAGCCACUGAUUAAAUAUCU 88 H44_56-77CUUCUGUUAGCCACUGAUUAAA 108 H44_61-82 UUCAGCUUCUGUUAGCCACUGA 89 H44_66-87AACUGUUCAGCUUCUGUUAGCC 109 H44_71-92 UGAGAAACUGUUCAGCUUCUGU 90 H44_76-97CUUUCUGAGAAACUGUUCAGCU 110 H44_81-102 UGUGUCUUUCUGAGAAACUGUU 91H44_86-107 GAAUUUGUGUCUUUCUGAGAAA 111 H44_91-112 CUCAGGAAUUUGUGUCUUUCUG112 H44_96-117 CAAUUCUCAGGAAUUUGUGUCU 113 H44_101-122GUUCCCAAUUCUCAGGAAUUUG 92 H44_106-127 AGCAUGUUCCCAAUUCUCAGGA 114H44_111-132 UAUUUAGCAUGUUCCCAAUUCU 93 H44_121-142 AUACCAUUUGUAUUUAGCAUGU94 H44_62-81 UCAGCUUCUGUUAGCCACUG 95

Experiments were performed in accordance with the condition and theprocedure of exon 45 (TEST EXAMPLE 1).

Experimental Results

The results are shown in FIGS. 9 and 10. These experiments revealedthat, when the antisense oligomers were designed at the 11th to the32nd, or the 26th to the 47th nucleotides from the 5′ end of exon 44 inthe human dystrophin gene, exon 44 skipping of these antisense oligomercould be caused with the same efficiency with that of the antisenseoligomer which is designed at the 62nd to the 81st nucleotides from the5′ end of exon 44.

Test Example 7 In Vitro Assay

Experiments were performed in accordance with the condition and theprocedure of exon 45 (TEST EXAMPLE 2).

Experimental Results

The results are shown in FIGS. 11, 12 and 18. This experiment revealedthat in RD cells, the oligomers PMO No. 24 and 26 (H44_25-45 (OH) andH44_27-47 (OH)) of the present invention caused exon 44 skipping withthe same efficiency as the antisense oligomer PMO No. 30 (H44_10-39(OH))(FIG. 11, 12). The oligomer PMO No. 26 of the present invention and theoligomer PMO No. 31 (H44_27-46(OH)), which is one base shorter than theoligomer PMO No. 26, were found to cause exon 44 skipping with the sameefficiency (FIG. 18). Furthermore, the oligomer PMO No. 26 of thepresent invention was found to cause exon 44 skipping with the sameefficiency as the oligomer PMO No. 33 (H44_27-47 (TEG)), which has adifferent end structure from the oligomer PMO No. 26 (FIG. 18).

Exon 50

According to the descriptions in EXAMPLES 30 to 39 below, various typesof PMO shown by PMO Nos. 34-38 and 41-45 in TABLE 15 were synthesized.The PMO synthesized was dissolved in water for injection (manufacturedby Otsuka Pharmaceutical Factory, Inc.). PMO Nos. 39 and 40 werepurchased from Gene Tools, LLC.

TABLE 15 PMO No. Sequence name Note SEQ ID NO: 34 H50_103-123(OH) 5′end: group (3) 96 35 H50_104-124(OH) 5′ end: group (3) 97 36H50_105-125(OH) 5′ end: group (3) 98 37 H50_106-126(OH) 5′ end: group(3) 99 38 H50_107-127(OH) 5′ end: group (3) 100 39 H50_90-114(GT)Sequence corresponding 101 to SEQ ID NO: 287 in Patent Document 4, 5′end: group (2) 40 H50_103-127(GT) Sequence corresponding 102 to SEQ IDNO: 175 in Patent Document 1, 5′ end: group (2) 41 H50_107-126(OH) 5′end: group (3) 119 42 H50_108-127(OH) 5′ end: group (3) 120 43H50_108-128(OH) 5′ end: group (3) 121 44 H50_109-129(OH) 5′ end: group(3) 122 45 H50_107-127(TEG) 5′ end: group (1) 100

Example 30 PMO. No. 34

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-{[(2S,6R)-6-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 3) was usedas the starting material.

ESI-TOF-MS Clcd.: 6861.8. Found: 6861.8.

Example 31 PMO. No. 35

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-{[(2S,6R)-6-(6-benzamideprine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 4) was usedas the starting material.

ESI-TOF-MS Clcd.: 6885.8. Found: 6885.9.

Example 32 PMO. No. 36

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylmolphorin-2-yl]methoxy]-4-oxo-butanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 2) was usedas the starting material.

ESI-TOF-MS Clcd.: 6925.9. Found: 6925.9.

Example 33 PMO. No. 37

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylmolphorin-2-yl]methoxy]-4-oxo-butanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 2) was usedas the starting material.

ESI-TOF-MS Clcd.: 6950.9. Found: 6950.9.

Example 34 PMO. No. 38

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylmolphorin-2-yl]methoxy]-4-oxo-butanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 2) was usedas the starting material.

ESI-TOF-MS Clcd.: 6990.9. Found: 6991.0.

Example 35 PMO. No. 41

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylmolphorin-2-yl]methoxy]-4-oxo-butanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 2) was usedas the starting material.

ESI-TOF-MS Clcd.: 6635.6. Found: 6635.0.

Example 36 PMO. No. 42

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylmolphorin-2-yl]methoxy]-4-oxo-butanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 2) was usedas the starting material.

ESI-TOF-MS Clcd.: 6635.6. Found: 6634.9.

Example 37 PMO. No. 43

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene resin(REFERENCE EXAMPLE 3) was used as the starting material.

ESI-TOF-MS Clcd.: 6965.9. Found: 6965.2.

Example 38 PMO. No. 44

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-{[(2S,6R)-6-(6-benzamidepurine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 4) was usedas the starting material.

ESI-TOF-MS Clcd.: 6949.9. Found: 6949.2.

Example 39 PMO. No. 45

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that1,12-dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoicacid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 5) was usedas the starting material.

ESI-TOF-MS Clcd.: 7342.2. Found: 7341.6.

Test Example 8 In Vitro Assay

Experiments were performed in accordance with the condition and theprocedure of exon 45 (TEST EXAMPLE 2), except that the RT-PCR wasperformed using the primers below in the concentrations of 0.1, 0.3 or 1μM.

Forward primer: (SEQ ID NO: 103) 5′-AACAACCGGATGTGGAAGAG-3′ Reverseprimer: (SEQ ID NO: 104) 5′-TTGGAGATGGCAGTTTCCTT-3′

Experimental Results

The results are shown in FIGS. 13 and 19. These experiments revealedthat in RD cells the oligomers PMO No. 38 (H50_107-127 (OH)) of thepresent invention caused exon 50 skipping with a higher efficiency thanthe antisense oligomer PMO No. 39 or 40 (H50_90-114 (GT), H50_103-127(GT)). Also, the experiments revealed that the oligomer PMO No. 38caused exon 50 skipping with a higher efficiency than the oligomer PMONo. 45 (H50_107-127 (TEG)), whose end structure is different from thatof the oligomer PMO No. 38 (FIG. 19).

Examination of Exon 44 Skipping In Vitro Assay Using Human FibroblastsTest Example 9

The exon 44 skipping activity was determined using GM05112 cells (humanDMD patient-derived fibroblasts with deletion of exon 45, CoriellInstitute for Medical Research). As a growth medium, there was usedDulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12)(Invitrogen Corp.) containing 10% FCS and 1% Penicillin/Streptmycin(P/S) (Sigma-Aldrich, Inc.) and the cells were cultured under conditionsof 37° C. and 5% CO₂.

The cells were cultured in T225 flask and the 2.5 mL of retrovirus(ZsGreen1 coexpression) expressing human derived MyoD (SEQ ID NO: 38)and a final concentration of 8 μg/mL of polybrene (Sigma-Aldrich, Inc.)were added to 35 mL of the growth medium. After incubation at 32° C. for2 days, the medium was exchanged to a fresh growth medium and incubationwas further continued at 37° C. for 3 days. ZsGreen1-positiveMyoD-transformed fibroblasts were collected by BD FACSAria Cell Sorter(BD Bioscience) and plated at 9×10⁴ cells/well into a collagen-coated24-well plate. The next day, the medium was replaced by adifferentiation medium (DMEM/F-12 containing 2% equine serum (InvitrogenCorp.), 1% P/S and ITS Liquid Media Supplement (Sigma, Inc.)). Themedium was exchanged every 2 to 3 days and incubation was continued todifferentiate into myotubes.

On the 7th day after the medium was changed to the differentiationmedium, the medium was replaced by a differentiation medium containing 6μM at a final concentration of Endo-Porter (Gene Tools), and 1, 3, 10 μMof the oligomers PMO No. 26 and 31 were added thereto at a finalconcentration. After the cells were incubated for 7 days, the cells werecollected to extract total RNA using RNeasy Mini Kit (QIAGEN). RT-PCRwas performed with 50 ng of the extracted total RNA using a QIAGENOneStep RT-PCR Kit. A reaction solution was prepared in accordance withthe protocol attached to the kit. An iCycler (manufactured by Bio-Rad)was used as a thermal cycler. The RT-PCR program used is as follows.

-   -   50° C., 30 mins: reverse transcription    -   95° C., 15 mins: thermal denaturation    -   [94° C., 1 mins; 60° C., 1 mins; 72° C., 1 mins]×35 cycles: PCR        amplification 72° C., 7 mins: final extension reaction

The nucleotide sequences of the forward primer and reverse primer usedfor RT-PCR are given below.

Forward primer: (SEQ ID NO: 36) 5′-GCTCAGGTCGGATTGACATT-3′ Reverseprimer: (SEQ ID NO: 37) 5′-GGGCAACTCTTCCACCAGTA-3′

The reaction product of RT-PCR above was separated by 2% agarose gelelectrophoresis and gel images were captured with an image analyzerImageQuant LAS 4000 mini (manufactured by FUJI Film). Using the attachedsoft, the polynucleotide level “A” of the band with exon 44 skipping andthe polynucleotide level “B” of the band without exon 44 skipping weremeasured. Based on these measurement values of “A” and “B”, the skippingefficiency was determined by the following equation:

Skipping efficiency (%)=A/(A+B)×100

Experimental Results

The result is shown in FIG. 20. These experiments revealed that inGM05112 cells the oligomers PMO No. 26 and 31 of the present inventioncaused exon 44 skipping with a high efficiency.

Test Example 10

A MyoD-transformed fibroblasts were prepared using GM05112 cells inaccordance with the procedure of TEST EXAMPLE 9, and the cells weredifferentiated into myotubes. Subsequently, the differentiation mediumwas replaced by a differentiation medium containing 6 μM at a finalconcentration of Endo-Porter (Gene Tools), and the oligomers PMO Nos. 26and 31 were added to the cells at a final concentration of 10 μM on the6th day after the medium was changed to the differentiation medium.After incubation for 14 days, the cells were collected by a scraperusing a cell lysis buffer RIPA buffer (manufactured by Pierce)containing a protease inhibitor cocktail Complete Mini (manufactured byRoche). The cell lysate were extracted from the cells by disrupting thecells by a ultrasonic crusher Bioruptor UCD-250 (Tosho Denki) andcollecting the supernatant after centrifugation. The proteinconcentrations were quantified using a Pierce BCA protein assay kit(Pierce). The absorbance of 544 nm of wavelength was detected using aplate reader Thermo Appliskan Type2001 (Thermo Electron).

The 3 μg of cell lysates were electrophoresed in acrylamide gel NuPAGENovex Tris-Acetate Gel 3-8% (manufactured by Invitrogen) and transferredonto a Immobilon-P membrane (manufactured by Millipore) using a semi-dryblotter. The transferred membrane was washed with PBS (PBST) containing0.1% Tween20 and blocked with PBST containing 5% Amersham ECL PrimeBlocking agent (GE Healthcare) in the refrigerator overnight. After themembrane was washed with PBST, the membrane was incubated in a solutionof anti-dystrophin antibody (manufactured by NCL-Dys1, Novocastra)50-fold diluted with Can Get Signal1 (manufactured by TOYOBO) at roomtemperature for 1 hour. After washing with PBST, the membrane wasincubated in a solution of peroxidase-conjugated goat-antimouse IgGantibody (170-6516, Bio-Rad) 2,500-fold diluted with Can Get Signal1(manufactured by TOYOBO) at room temperature for 10 minutes. Afterwashing with PBST, the membrane was stained with ECL Plus WesternBlotting Detection System (GE Healthcare). The chemiluminescence of thedystrophin protein deleted exon 44-45 was detected by lumino imageanalyzer ImageQuant LAS 4000 mini (FUJI Film).

Experimental Results

The results of Western blotting are shown in FIG. 21. In FIG. 21, thearrowhead represents a band of dystrophin protein of which theexpression was confirmed. This experiment reveals that the oligomers PMONo. 26 and 31 of the present invention induced expression of dystrophinproteins in GM05112 cells.

Study of Exon 50 Skipping In Vitro Assay Using Human Fibroblasts TestExample 11

MyoD-transformed fibroblasts were prepared using GM05112 cells todifferentiate into myotubes in accordance with the procedure of TESTEXAMPLE 9.

Subsequently, the differentiation medium was replaced by adifferentiation medium containing 6 μM of Endo-Porter (Gene Tools), anda oligomer PMO No. 38 was added thereto at a final concentration of 0.1,0.3, 1, 3, 10 μM on the 12th day after the medium was changed to thedifferentiation medium. After incubation for 2 days, the cells werecollected. Total RNA was extracted from the cells, RT-PCR was performedand the skipping efficiency was determined in accordance with theprocedure of TEST EXAMPLE 9, except that the nucleotide sequences of theforward primer and reverse primer given below were used for RT-PCR.

Forward primer: (SEQ ID NO: 103) 5′-AACAACCGGATGTGGAAGAG-3′ Reverseprimer: (SEQ ID NO: 104) 5′-TTGGAGATGGCAGTTTCCTT-3′

Experimental Results

The result of RT-PCR is shown in FIG. 22 and the result of skippingefficiency is shown in FIG. 23. These experiments revealed that inGM05112 cells the oligomer PMO No. 38 of the present invention causedexon 50 skipping with a high efficiency and the value of EC₅₀ was 1.3μM.

Test Example 12

Experiments for skipping were performed in accordance with the conditionand the procedure of TEST EXAMPLE 11, except that 11-0627 cells (humanDMD patient derived fibroblasts with duplication of exons 8-9, NationalCenter of Neurology and Psychiatry neuromuscular disorder researchresource repository) were used and the oligomer PMO No. 38 was added ata final concentration of 0.1, 1, 10 μM.

Experimental Results

The result of RT-PCR is shown in FIG. 26 and the skipping efficiency isshown in FIG. 27. These experiments revealed that in 11-0627 cells theoligomer PMO No. 38 of the present invention caused exon 50 skippingwith a high efficiency.

Test Example 13

pLVX-MyoD-ZsGreen1 Lentivirus Preparation

pLVZ-puro (8120 bp, Clontech) was linearized by deleting 1164 bpnucleotides which is located on from XhoI site in the multicloning site(at 2816) to the site (at 3890) adjacent to the 3′ end of Puromycinresistant gene coding region to prepare a linearized vector.Subsequently, the nucleotide sequences (2272 bp) which encodes humanMyoD gene, IRES sequence, ZsGreen1 gene was integrated into thelinearized vector in turn and then the lentivirus expression vectorpLVX-MyoD-ZsGreen1 (9210 bp) was prepared.

Lenti-X 293T cells were plated onto 10 cm collagen coated dish inaccordance with the protocol attached to Lenti-X HTX Packaging System(Clontech). Lentivirus expression vector and packaging vector weretransfected into fibroblasts three days before infection. After fourhours, the medium was exchanged and the cells were incubated for threedays without exchanging medium. On the day of infection, the culturesupernatant was collected as a virus solution (about 9 mL for 10 cmdish). The culture supernatant was filtrated by cellstrainer (40 μm) andthen centrifuged by 500×g, 10 min. This supernatant was concentrated inaccordance with the protocol attached to Lenti-X Concentrator (Clontech)and then dissolved in DMEM/F12 medium to ten times the concentration ofthe collected culture supernatant. This solution was used as a virussolution.

Virus Infection into Fibroblasts

GM04364 cells (human DMD patient derived fibroblasts deleted exons51-55, Coriell Institute for Medical Research) were plated on acollagen-coated 24-well plate by 3×10⁴/well by the day of infection. Onthe day of infection, 400 μL of the differentiation medium, 100 μL ofthe virus solution, and 8 μg/mL of polybrene at a final concentrationper well were added. The day after infection, the medium containingvirus was exchanged into 500 μL of the differentiation medium. Thedifferentiation medium was exchanged every 2 or 3 days and the cellswere incubated for 12 days to induce differentiation into myotubes.

On the 12th day after the medium was exchanged into the differentiationmedium, the medium was replaced by a differentiation medium containing 6μM Endo-Porter (Gene Tools) at a final concentration, and 0.1, 0.3, 1,3, 10 μM of the oligomer PMO No. 38 was added thereto at a finalconcentration. After incubation for 2 days, the cells were collected.The skipping efficiency was determined in accordance with the procedureof TEST EXAMPLE 11, except that the nucleotide sequences of the forwardprimer and reverse primer given below were used for RT-PCR.

Forward primer: (SEQ ID NO: 103) 5′-AACAACCGGATGTGGAAGAG-3′ Reverseprimer: (SEQ ID NO: 70) 5′-CTGCCGGCTTAATTCATCAT-3′

Experimental Results

The result of RT-PCR is shown in FIG. 28 and the skipping efficiency isshown in FIG. 29. These experiments revealed that in GM04364 cells theoligomer PMO No. 38 of the present invention caused exon 50 skippingwith a high efficiency.

Study of Exon 55 Skipping In Vitro Assay Using Human Fibroblasts TestExample 14

Experiments were performed in accordance with the condition and theprocedure of TEST EXAMPLE 11, except that the oligomers PMO No. 14 and21 were used and the RT-PCR was performed using the primers below.

Forward primer: (SEQ ID NO: 69) 5′-CATGGAAGGAGGGTCCCTAT-3′ Reverseprimer: (SEQ ID NO: 70) 5′-CTGCCGGCTTAATTCATCAT-3′

Experimental Results

The result of RT-PCR is shown in FIG. 24 and the skipping efficiency isshown in FIG. 25. These experiments revealed that in GM05112 cells theoligomers PMO No. 14 and 21 of the present invention caused exon 55skipping with a high efficiency and the value of EC₅₀ was 3.5 μM and 7.5μM, respectively.

Test Example 15

Experiments were performed in accordance with the condition and theprocedure of TEST EXAMPLE 13, except that the 04-035 cells (human DMDpatient derived cells with single deletion of exon 54, National Centerof Neurology and Psychiatry neuromuscular disorder research resourcerepository) were used and 1, 3, 10 μM of the oligomers PMO No. 14 and 21at a final concentration were added and the RT-PCR was performed usingthe primers below.

Forward primer: (SEQ ID NO: 69) 5′-CATGGAAGGAGGGTCCCTAT-3′ Reverseprimer: (SEQ ID NO: 70) 5′-CTGCCGGCTTAATTCATCAT-3′

Experimental Results

The result of RT-PCR is shown in FIG. 30 and the skipping efficiency isshown in FIG. 31. These experiments revealed that in human DMD patientderived cells with single deletion of exon 54, the oligomers PMO No. 14and 21 of the present invention caused exon 55 skipping with a highefficiency.

INDUSTRIAL APPLICABILITY

Experimental results in TEST EXAMPLES demonstrate that the oligomers ofthe present invention caused exon skipping with a markedly highefficiency in both RD cells and DMD patients derived cells.

Therefore, the oligomers of the present invention are extremely usefulfor the treatment of DMD.

Sequence Listing Free Text

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Sequence Listing:

1. An antisense oligomer which causes skipping of the 55th exon in thehuman dystrophin gene, consisting of a nucleotide sequence complementaryto any one of the nucleotide sequences consisting of the 10th to the31st, the 10th to the 32nd, the 11th to the 31st, the 11th to the 32nd,the 13th to the 34th, the 13th to the 35th, the 13th to the 36th, 14thto the 35th, the 14th to the 36th, the 15th to the 35th, the 15th to the36th, the 16th to the 34th, the 16th to the 35th, and the 16th to the36th nucleotides, from the 5′ end of the 55th exon in the humandystrophin gene. 2-5. (canceled)
 6. The antisense oligomer according toclaim 1, consisting of the nucleotide sequence shown by any one selectedfrom the group consisting of the 160th to the 181st, the 159th to the181st, the 160th to the 180th, the 159th to the 180th, the 157th to the178th, the 156th to the 178th, the 155th to the 178th, the 156th to the177th, the 155th to the 177th, the 156th to the 176th, the 155th to the176th, the 157th to the 175th, the 156th to the 175th, and the 155th tothe 175th nucleotides of SEQ ID NO:
 5. 7-12. (canceled)
 13. Theantisense oligomer according to claim 1, which is an oligonucleotide.14. The antisense oligomer according to claim 13, wherein the sugarmoiety and/or the phosphate-binding region of at least one nucleotideconstituting the oligonucleotide is modified.
 15. The antisense oligomeraccording to claim 14, wherein the sugar moiety of at least onenucleotide constituting the oligonucleotide is a ribose in which the2′-OH group is replaced by any one selected from the group consisting ofOR, R, R′OR, SH, SR, NH₂, NHR, NR₂, N₃, CN, F, Cl, Br and I (wherein Ris an alkyl or an aryl and R′ is an alkylene).
 16. The antisenseoligomer according to claim 14, wherein the phosphate-binding region ofat least one nucleotide constituting the oligonucleotide is any oneselected from the group consisting of a phosphorothioate bond, aphosphorodithioate bond, an alkylphosphonate bond, a phosphoramidatebond and a boranophosphate bond.
 17. The antisense oligomer according toclaim 1, which is a morpholino oligomer.
 18. The antisense oligomeraccording to claim 17, which is a phosphorodiamidate morpholinooligomer.
 19. The antisense oligomer according to claim 17, wherein the5′ end is any one of the groups of chemical formulae (1) to (3) below:


20. A pharmaceutical composition for the treatment of musculardystrophy, comprising as an active ingredient the antisense oligomeraccording to claim 1, or a pharmaceutically acceptable salt or hydratethereof.
 21. A method of treating muscular dystrophy, comprisingadministering to a patient in need thereof a therapeutically effectiveamount of the antisense oligomer according to claim
 1. 22. A method oftreating muscular dystrophy, comprising administering to a patient inneed thereof a therapeutically effective amount of pharmaceuticalcomposition of claim 20.